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WO2014183023A1 - Utilisation de la plexine-a4 comme biomarqueur et cible thérapeutique pour la maladie d'alzheimer - Google Patents

Utilisation de la plexine-a4 comme biomarqueur et cible thérapeutique pour la maladie d'alzheimer Download PDF

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Publication number
WO2014183023A1
WO2014183023A1 PCT/US2014/037479 US2014037479W WO2014183023A1 WO 2014183023 A1 WO2014183023 A1 WO 2014183023A1 US 2014037479 W US2014037479 W US 2014037479W WO 2014183023 A1 WO2014183023 A1 WO 2014183023A1
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seq
plxna4
subject
nucleic acid
complement
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PCT/US2014/037479
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English (en)
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Lindsay A. Farrer
Gyungah JUN
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Trustees Of Boston University
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Priority to US14/889,565 priority Critical patent/US20160186263A1/en
Publication of WO2014183023A1 publication Critical patent/WO2014183023A1/fr

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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/34—Allele or polymorphism specific uses
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates generally to assays, methods, and systems for diagnosing Alzheimer's disease (AD).
  • the invention further relates to methods and compositons for treatment of AD.
  • Alzheimer's disease is a leading cause of dementia in the elderly, affecting 5-10% of the population over the age of 65 years (A Guide to Understanding Alzheimer's Disease and Related Disorders, Jorm, ed., New York University Press, New York, 1987).
  • Alzheimer's disease the parts of the brain essential for cognitive processes such as memory, attention, language, and reasoning degenerate, robbing victims of much that makes us human, including independence.
  • onset can first be seen in middle age, but more commonly, symptoms appear from the 65 and onwards.
  • Alzheimer's disease today affects 4-5 million Americans, with slightly more than half of these people receiving care at home, while the others are in many different health care institutions.
  • the prevalence of Alzheimer's disease and other dementias doubles every 5 years beyond the age of 65, and recent studies indicate that nearly 50% of all people age 85 and older have symptoms of Alzheimer's disease (1999 Progress Report on Alzheimer's Disease, National Institute on Aging/National Institute of Health). 13% (33 million people) of the total population of the United States are age 65 and older, and this percentage will climb to 20%> by the year 2025 (1999 Progress Report on Alzheimer's Disease).
  • Alzheimer's disease also puts a heavy economic burden on society.
  • a recent study estimated that the cost of caring for one Alzheimer's disease patient with severe cognitive impairments at home or in a nursing home, is more than $47,000 per year (A Guide to Understanding Alzheimer's Disease and Related Disorders).
  • the annual economic toll of Alzheimer's disease in the United States in terms of health care expenses and lost wages of both patients and their caregivers is estimated at $80 to $100 billion (1999 Progress Report on Alzheimer's disease).
  • AD Alzheimer disease
  • methods and assays comprising PLXNA4.
  • methods of treating Alzheimer disease (AD) by inhibiting phosphorylation of tau inhibitor via inhibition of PLXNA4 or one of its ligands including, but not limited to, SEMA3.
  • PLXNA4 can be used as a biomarker by measuring the expression levels of the full-length and shorter PLXNA4 isoforms in serum or by genotyping PLXNA4 single nucleotide polymorphisms (SNPs) that are genetically associated with AD including, but not limited to, rs277470, rs277472, rs277473, rs277474, rs277476, rs277477, rs277478, rs277479, rs277480, rs277481, rs277483, rs277484, rsl0234979, rs9641933, rsl0225863, rs7799929, rs9656410, rsl 1764790, rs4731860, rsl 1763817, rsl3231950, rsl 1773243, rsl7166338, rsl
  • SNPs single nucleotide polymorphisms
  • NIA-LOAD National Institute on Aging Late Onset Alzheimer Disease
  • PLXNA4 or its binding partners can be used as novel drug targets for AD, as well as markers in assays for classifying and identifying subjects at risk for Alzheimer's disease, such as serum-based assays.
  • assays using PLXNA4 as a biomarker in serum are assays using PLXNA4 variants for predictive testing or stratification of subjects in clinical trials and for treatment purposes, and PLXNA4 variants, and their binding partners, as targets in methods for treatment of Alzheimer's disease.
  • the assay and method comprise (a) transforming a biological sample from the subject into at least one detectable target loci for a nucleic acid polymorphism, wherein the target locus is selected from: SNP rs277472, SNP rsl0236235, and SNP rsl 1761937; and (b) detecting presence or absence of at least one (e.g., one, two, three or more) AD risk associated SNPs from the the at least one detectable target loci.
  • AD risk associated SNPs include A/A or AJC SNP rs277472, T/T or T/C SNP rsl0236235, and C/C or C/A SNP rsl 1761937.
  • Another aspect of the assays and methods for determining an increased risk for developing AD in a subject include measuring the amount of at least one gene associated with the AD risk associated SNPs described herein in a biological sample from the subject, and then comparing the measured amount of the gene to a reference amount.
  • at least the amount of PLXNA4 gene expression products e.g., nucleic acid or protein
  • SNP rs277472, SNP rsl0236235, or SNP rsl 1761937 is measured in a biological sample of a subject and compared to a reference level.
  • expression of full length isoform 1 (TS1) or shorter isoform 3 (TS3) of PLXNA4 gene expression product is measured.
  • the amount of the PLXNA4 gene expression products can be higher by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%), at least about 70%>, at least about 80%>, at least about 90%>, or at least about 95%, about 98%, about 99% or 100%, or hihgher than the reference level.
  • the reference amount can be that measured in a normal healthy subject with no genetic susceptibility for AD.
  • a normal healthy subject that is not a carrier of any of the AD risk associated alleles described herein or is not diagnosed with any forms of AD such as early-onset autosomal-dominant AD, or any neurodegenerative disorders.
  • the reference amount can be from a control sample, a pooled sample of control individuals or a numeric value or range of values based on the same.
  • the invention provides methods or assays for determining if an individual is in need of AD treatment or prevention, comprising the steps of determining if the subject carries any of the SNPs selected from the group consisting of: (i) SNP1 genotype A/A or A/C (or T/T or T/G in the complement) of SEQ ID NO: 1, wherein SNP1 is identified by rs277472 on SEQ ID NO: 1, wherein SEQ ID NO.
  • SNP1 is a portion of genomic nucleic acid sequence of plexin A4 (PLXNA4); (ii) SNP2 genotype T/T or T/C (or A/A or A/C in the complement) of SEQ ID NO: 1, wherein SNP2 is position 132,006,366 of SEQ ID NO: 1 identified by rs 10236235, wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; (iii) SNP3 genotype C/C or C/A (or G/G or G/T in the complement) of SEQ ID NO: 1, wherein SNP3 identified by rsl 1761937 on SEQ ID NO: 1, wherein the SEQ ID NO.
  • PLXNA4 plexin A4
  • the subject can further be administered a treatment or prevention intervention to treat AD symptoms or inhibit development of AD symptoms.
  • treatment of prevention interventions include, but are not limited to life style advise, including e.g., prescribing an aerobic exercise regime, dietary advise, including increase in intake of omega-3 fatty acids or reduction of sugar or cholesterol rich food intake to lower cholesterol, and administering pharmaceutical agents effective in prevention or treatment of AD.
  • the treatment includes administering a therapeutically effective amount of a TS1 PLXNA4 inhibitory agent.
  • a further aspect of the invention provides a computer implemented system for determining presence or absence of alleles associated with an increased risk of a subject for developing late onset Alzheimer's disease (AD).
  • the system comprises (a) a determination module configured to identify and detect at least one single nucleotide polymorphism (SNP) in a biological sample of a subject, wherein the SNP is selected from: (i) (i) SNP1 genotype A/A or A/C (or T/T or T/G in the complement) of SEQ ID NO: 1, wherein SNP1 is identified by rs277472 on SEQ ID NO: 1, wherein SEQ ID NO.
  • SNP single nucleotide polymorphism
  • SNP1 is a portion of genomic nucleic acid sequence of plexin A4 (PLXNA4); (ii) SNP2 genotype T/T or T/C (or A/A or A/C in the complement) of SEQ ID NO: 1, wherein SNP2 is position 132,006,366 of SEQ ID NO: 1 identified by rs 10236235, wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; (iii) SNP3 genotype C/C or C/A (or G/G or G/T in the complement) of SEQ ID NO: 1, wherein SNP3 identified by rsl 1761937 on SEQ ID NO: 1, wherein the SEQ ID NO.
  • PLXNA4 plexin A4
  • a portion of genomic nucleic acid sequence of PLXNA4 is a portion of genomic nucleic acid sequence of PLXNA4; and (iv) any combinations thereof;
  • a storage module configured to store output data from the determination module;
  • a computing module adapted to identify whether at least one AD risk associated SNP is present or absent in the output data stored on the storage module; and
  • a display module for displaying if any of the AD risk associated SNP was identified or not.
  • the display module can display the detected alleles.
  • Yet another aspect of the invention relates to a pharmaceutical composition and methods for treating AD in a subject.
  • the method comprises administering to the subject a pharmaceutically acceptable composition comprising a TS1 PLXNA4 inhibitory agent.
  • the method further comprises diagnosing the individual as having or at risk of AD prior to administering the agent.
  • the diagnosing can be performed, e.g, using the method of determining the level of PLXNA4 (e.g., isoform TS1 or TS3) or by detecting the presence or absence of any one or more of the AD associated SNPs disclosed herein.
  • a still yet another aspect of the invention relates to a method for determining if a subject is in need of treatment or prevention for AD.
  • the method comprises the steps of: (a) transforming at least one (e.g., one, two, or three) nucleic acid polymorphism in a locus in a biological sample from the subject into at least one detectable target, wherein the locus is selected from SNP rs277472, SNP rsl0236235, or SNP rsl 1761937; and (b) detecting presence or absence of at least one AD risk associated SNP from the at least one detectable target, wherein detection of the presence of at least one AD risk associated allele is indicative of the subject in need for treatment or prevention for AD.
  • the method further comprises administering a treatment or preventive intervention to the subject, if presence of at least one AD risk-associated SNP is detected.
  • the disclosure provides a assay ofr identifying a subject having or at risk for Alzheimer's disease.
  • the method comprising measuring or quantifying the expression level or amount of one or both of TS1 andTS3 PLXNA4 transcripts (e.g., mR A) in a biological sample obtained from the subjet and identifying the subject as having or at risk for Alzheimer's disease if the expression level or amount of one or both of TS1 and TS3 PLXNA4 transcript is increased relative to a reference value.
  • TS1 andTS3 PLXNA4 transcripts e.g., mR A
  • Figs. 1A-1F show genetic findings in the PLXNA4 region.
  • Most significant SNPs in the FHS (rs277472) and NIA-LOAD (rsl 1761937) datasets are indicated by purple diamonds.
  • P-values are expressed as -loglO(P) (y-axis) for every tested SNP ordered by chromosomal location (x-axis).
  • Genomic structure was determined using the NCBI database (Build 37.1). Three validated transcripts (TS1, TS2, and TS3) are shown. Top association signals are highlighted in pink for the FHS dataset and in yellow for the NIA-LOAD dataset. The gene structure and reading frame are indicated with a pink arrow. Exons are denoted with vertical bars on the arrow.
  • rs277472 ID
  • IE rsl 1761937/rsl0236235
  • SEMA sema_plexinAl interacting module
  • PSI plexin repeat
  • IPT three repeats of the binding domains of plexins and cell surface receptors
  • IPT PCSR binding domain of plexins and cell surface receptors (PCSR) and related proteins
  • TM transmembrane region
  • CYTO cytoplasmic domain.
  • Fig. ID are (i) GGTCCTCGCCTCC (SEQ ID NO: 2) and (ii) GGTCCTAGCCTCC (SEQ ID NO: 3); in Fig. IE are (i)
  • GTTTGCCGTGTCG (SEQ ID NO: 4); (i) GTTTGCTGTGTCG (SEQ ID NO: 5); (iii) TCCCAAACTCCTG (SEQ ID NO: 6); and (iv) TCCCAACCTCCTG (SEQ ID NO: 7).
  • FIGS. 2A-2C show PLXNA4 isoforms in tau phosphorylation.
  • TS1 or TS2 and TS3 full-length
  • TS2 and TS3 the full-length
  • TS2 and TS3 the shorter isoforms
  • pcDNA3.1 empty vectors
  • Whole cell lysates were blotted with AT8, total tau, actin and Myc.
  • results for the TS2 and TS3 isoforms were similar but ony those for TS3 are shown.
  • FIGS. 3A-3B show RNA expression of PLXNA4 isoforms.
  • 3B isoforms.
  • FIGS. 4A-4E show genetic findings in the PLXNA4 region.
  • PLXNA4 was determined using the NCBI database (Build 37.1).
  • SEMA sema_plexinAl interacting module (exon 1-4 in TSl and exon 1-3 in TS2 and TS3); PSI: plexin repeat (exon 4-11 in TSl); IPT: three repeats of the binding domains of plexins and cell surface receptors (exon 11-17 n TSl); PCSR: binding domain of plexins and cell surface receptors (PCSR) and related proteins (exon 17-19 in TSl); TM: transmembrane region (exon 19 in TSl); CYTO: cytoplasmic domain (exon 20-31 in TSl).
  • FIG. 5 shows linkage disequilibrium (LD, D') of top ranked SNPs.
  • LD was calculated in 1000 genomes data from CEU for Caucasian, AFR for African American, and ASN for Japanese populations.
  • Top-ranked SNPs from the PLXNA4 gene rs277470, rs277472, and rs277484 in FHS; rsl2539196 in NIA-LOAD; rsl0273901 in ADGC-EA; rs75460865 in ADGC-AA; rs 13232207 in ADGC-JPN.
  • the top SNP from the ADGC-AA was monomorphic in both EUR and ASN populations.
  • top-ranked SNPs (rs 10273901, rs75460865, rs277470, rs277472, and rs277484) from the Caucasian and African American samples were monomorphic in the ASN sample.
  • the top ranked SNPs are located in the SEMA domain, except rsl0273901 and rsl3232207 which are located in the cytoplasmic domain.
  • Embodiments of the various aspects disclosed herein are generally related to assays, methods and systems for identifying a subject with an increased risk for late -onset AD.
  • the assays, methods and systems are directed to detection of single nucleotide polymorphisms (SNPs) associated with late-onset AD in a biological sample of a subject.
  • the assays, methods and systems are directed to SNPs
  • Another aspect of the invention is directed to methods and
  • compositions for therapeutic treatment of AD e.g., by administering aTSl PLXNA4 inhibitory agent to a subject diagnosed with or at risk of AD.
  • the disclosure provides a method for inhibiting progression of AD.
  • the method comprising administering having or at risk for Alzheimer's disease a
  • the method further comprises assaying a biological sample from the subjet for the presence or absence of one or more AD risk associated SNPs before onset of said administering.
  • the disclosure also provides a method for inhibiting or reducing neurofibrillary tangles in the brain.
  • the method comprising administering to a subject having or at risk for having neurofibrillary tangles in the brain a therapeutically effective amount of a TS 1 PLXNA4 inhibitory agent.
  • the method further comprises assaying a biological sample from the subjet for the presence or absence of one or more AD risk associated SNPs before onset of said administering.
  • the disclosure provides a method for inhibiting or reducing tau phosphorylation in the brain.
  • the method comprising administering to a subject a therapeutically effective amount of a TSl PLXNA4 inhibitory agent.
  • the method further comprises assaying a biological sample from the subjet for the presence or absence of one or more AD risk associated SNPs before onset of said administering.
  • a TS 1 PLXNA4 inhibitory agent refers to an agent that can inhibit expression and/or activity of the TS1 PLXNA4 isoform.
  • a TS1 PLXNA4 inhibitory agent can be specific for the TS1 isoform.
  • a TS1 PLXNA4 inhibitory agent also has some activity against other PLXNA4 isoforms, such as the TS2 and TS2 PLXNA4 isoforms, as described herein.
  • agent or “compound” as used herein, in regard to, for example, a TS1 PLXNA4 inhibitory agent, refers to a chemical entity or biological product, or combination of chemical entities or biological products, administered to a subject to treat or prevent or control a disease or condition.
  • the chemical entity or biological product is preferably, but not necessarily a low molecular weight compound, but may also be a larger compound, or any organic or inorganic molecule effective in the given situation, including modified and unmodified nucleic acids such as antisense nucleic acids, R Ai, such as siR A or shRNA, peptides, peptidomimetics, receptors, ligands, and antibodies, aptamers, polypeptides, nucleic acid analogues or variants thereof.
  • modified and unmodified nucleic acids such as antisense nucleic acids, R Ai, such as siR A or shRNA, peptides, peptidomimetics, receptors, ligands, and antibodies, aptamers, polypeptides, nucleic acid analogues or variants thereof.
  • R Ai such as siR A or shRNA
  • peptides peptidomimetics
  • receptors receptors
  • ligands and antibodies
  • aptamers polypeptides
  • Agents can be selected from a group comprising: chemicals; small molecules; nucleic acid sequences; nucleic acid analogues; proteins; peptides; aptamers; antibodies; or fragments thereof.
  • a nucleic acid sequence can be RNA or DNA, and can be single or double stranded, and can be selected from a group comprising; nucleic acid encoding a protein of interest, oligonucleotides, nucleic acid analogues, for example peptide-nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), locked nucleic acid (LNA), modified RNA (mod- RNA) etc.
  • PNA peptide-nucleic acid
  • pc-PNA pseudo-complementary PNA
  • LNA locked nucleic acid
  • modified RNA mod- RNA
  • nucleic acid sequences include, for example, but are not limited to, nucleic acid sequence encoding proteins, for example that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but are not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc.
  • a protein and/or peptide or fragment thereof can be any protein of interest, for example, but are not limited to: mutated proteins; therapeutic proteins and truncated proteins, wherein the protein is normally absent or expressed at lower levels in the cell.
  • Proteins can also be selected from a group comprising; mutated proteins, genetically engineered proteins, peptides, synthetic peptides, recombinant proteins, chimeric proteins, antibodies, midibodies, minibodies, triabodies, humanized proteins, humanized antibodies, chimeric antibodies, modified proteins and fragments thereof.
  • the agent can be intracellular within the cell as a result of introduction of a nucleic acid sequence into the cell and its transcription resulting in the production of the nucleic acid and/or protein modulator of, for example, a TS1 PLXNA4 transcript, within the cell.
  • the agent is any chemical, entity or moiety, including without limitation synthetic and naturally-occurring non- proteinaceous entities.
  • the agent is a small molecule having a chemical moiety.
  • chemical moieties included unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof.
  • Agents can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds.
  • the TS1 PLXNA4 inhibitory agent is an oligonucleotide. In some embodiments, the TS 1 PLXNA4 inhibitory agent is an anti-miR, antagomir, antisense oligonucleotide, ribozyme, aptamer, siRNA, shRNA, or RNAi agent.
  • the TS 1 PLXNA4 inhibitory agent is an antisense oligonucleotide.
  • oligonucleotides can hybridize to a complementary target sequence and prevent access of the translation machinery to the target RNA transcript, thereby preventing protein synthesis.
  • the single- stranded oligonucleotide can also hybridize to a complementary RNA and the RNA target can be subsequently cleaved by an enzyme such as RNase H and thus preventing translation of target RNA.
  • the single-stranded oligonucleotide can modulate the expression of a target sequence via RISC mediated cleavage of the target sequence, i.e., the single-stranded oligonucleotide acts as a single-stranded RNAi agent.
  • a "single-stranded RNAi agent" as used herein, is an RNAi agent which is made up of a single molecule.
  • a single-stranded RNAi agent can include a duplexed region, formed by intra- strand pairing, e.g., it can be, or include, a hairpin or pan-handle structure.
  • the TS1 PLXNA4 inhibitory agent is RNA-interference or RNA interference molecule, including, but not limited to double-stranded RNA, such as siRNA, double-stranded DNA or single-stranded DNA.
  • an anti-miR- 130/301 agent is a single-stranded RNA (ssRNA), a form of RNA endogenously found in eukaryotic cells as the product of DNA transcription.
  • Cellular ssRNA molecules include messenger RNAs (and the progenitor pre-messenger RNAs), small nuclear RNAs, small nucleolar RNAs, transfer RNAs and ribosomal RNAs.
  • Double-stranded RNA induces a size-dependent immune response such that dsRNA larger than 30bp activates the interferon response, while shorter dsRNAs feed into the cell's endogenous RNA interference machinery downstream of the Dicer enzyme.
  • dsRNA Double-stranded RNA
  • Numerous specific siRNA molecules have been designed that have been shown to inhibit gene expression (Ratcliff et al. Science 276: 1558-1560, 1997; Waterhouse et al.
  • siRNA molecules have been shown to inhibit, for example, HIV-1 entry to a cell by targeting the host CD4 protein expression in target cells thereby reducing the entry sites for HIV-1 which targets cells expressing CD4 (Novina et al. Nature Medicine, 8:681-686, 2002). Short interfering RNA have further been designed and successfully used to silence expression of Fas to reduce Fas-mediated apoptosis in vivo (Song et al. Nature Medicine 9:347-351, 2003).
  • This modification has important regulatory effects on gene expression, especially when involving CpG-rich areas known as CpG islands, located in the promoter regions of many genes. While almost all gene-associated islands are protected from methylation on autosomal chromosomes, extensive methylation of CpG islands has been associated with transcriptional inactivation of selected imprinted genes and genes on the inactive X-chromosomes of females. Aberrant methylation of normally unmethylated CpG islands has been documented as a relatively frequent event in immortalized and transformed cells and has been associated with transcriptional inactivation of defined tumor suppressor genes in human cancers. In this last situation, promoter region hypermethylation stands as an alternative to coding region mutations in eliminating tumor suppression gene function (Herman, et al.). The use of siRNA molecules for directing methylation of a target gene is described in U.S. Provisional Application No. 60/447,013, filed Feb. 13, 2003, referred to in U.S. Patent Application Publication No. 20040091918.
  • RNA interference does not have to match perfectly to its target sequence.
  • the 5' and middle part of the antisense (guide) strand of the siRNA is perfectly complementary to the target nucleic acid sequence.
  • the RNA interference-inducing molecule functioning as TS1 PLXNA4 inhibitory agent includes RNA molecules that have natural or modified nucleotides, natural ribose sugars or modified sugars and natural or modified phosphate backbone. Accordingly, the RNA interference-inducing molecules functioning as anti-miR-130/301 agen includes, but are not limited to, unmodified and modified double stranded (ds) RNA molecules including short-temporal RNA (stRNA), small interfering RNA (siRNA), short-hairpin RNA (shRNA), microRNA (miRNA), and double-stranded RNA (dsRNA), (see, e.g. Baulcombe, Science 297:2002-2003, 2002).
  • ds double-stranded RNA
  • the dsRNA molecules e.g. siRNA
  • the siRNA molecules do not include RNA molecules that comprise ssRNA greater than about 30-40 bases, about 40-50 bases, about 50 bases or more.
  • the siRNA molecules have a double stranded structure.
  • the siRNA molecules are double stranded for more than about 25%, more than about 50%, more than about 60%, more than about 70%, more than about 80% or more than about 90% of their length.
  • Anti-miRs including hairpin miRNA inhibitors, are described in detail in
  • a person of ordinary skill in the art can select a sequence from the database for a desired miRNA and design an inhibitor useful for the compositions and methods disclosed herein.
  • Anti-miRs can be used to efficiently silence endogenous miRNAs by forming duplexes comprising the anti- miR and endogenous miRNA, thereby preventing miRNA-induced gene silencing.
  • the TS1 PLXNA4 inhibitory agent is an antagomir.
  • Antagomirs are oligonucleotide anti-miRs that harbor various modifications for RNAse protection and pharmacologic properties, such as enhanced tissue and cellular uptake. They differ from normal RNA by, for example, complete 2'-O-methylation of sugar,
  • antagomir comprises a 2'-0-methylmodification at all nucleotides, a cholesterol moiety at 3 '-end, two phsophorothioate intersugar linkages at the first two positions at the 5 '-end and four phosphorothioate linkages at the 3 '-end of the molecule.
  • Antagomirs can be used to efficiently silence endogenous miRNAs by forming duplexes comprising the antagomir and endogenous miRNA, thereby preventing miRNA-induced gene silencing.
  • An example of antagomir-mediated miRNA silencing is the silencing of miR-122, described in Krutzfeldt et al, Nature, 2005, 438: 685-689, which is expressly incorporated by reference herein in its entirety.
  • the TS1 PLXNA4 inhibitory agent is ribozyme.
  • the anti-miR-130/301 agent is ribozyme that cleaves the target microRNA.
  • Ribozymes are oligonucleotides having specific catalytic domains that possess endonuclease activity (Kim and Cech, Proc Natl Acad Sci U S A. 1987 Dec;84(24):8788-92; Forster and Symons, Cell. 1987 Apr 24;49(2):211-20). At least six basic varieties of naturally-occurring enzymatic RNAs are known presently. In general, enzymatic nucleic acids act by first binding to a target RNA.
  • Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA.
  • the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
  • Ribozymes can be designed as described in Int. Pat. Appl. Publ. No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, each specifically incorporated herein by reference, and synthesized to be tested in vitro and in vivo, as described therein.
  • transcription factors recognize their relatively short binding sequences, even in the absence of surrounding genomic DNA, short oligonucleotides bearing the consensus binding sequence of a specific transcription factor can be used as tools for manipulating gene expression in living cells.
  • This strategy involves the intracellular delivery of such "decoy oligonucleotides", which are then recognized and bound by the target factor. Occupation of the transcription factor's DNA-binding site by the decoy renders the transcription factor incapable of subsequently binding to the promoter regions of target genes. Decoys can be used as therapeutic agents, either to inhibit the expression of genes that are activated by a transcription factor, or to up-regulate genes that are suppressed by the binding of a transcription factor.
  • the anti-miR-130/301 agent is a decoy oligonucleotide.
  • the TS 1 PLXNA4 inhibitory agent comprises a sequence substantially complimentary to at least 15 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) contiguous nucleotides of SEQ ID NO: 1.
  • the TS 1 PLXNA4 inhibitory agent comprises a sequence substantially identical to at least 15 (e.g., 15, 16, 17, 18, 19, 20., 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) contiguous nucleotides of SEQ ID NO: 1 [0043]
  • the TS1 PLXNA4 inhibitory agent comprises a nucleotide sequence substantially complementary to: (i) GGTCCTCGCCTCC (SEQ ID NO: 2); (ii) GGTCCTAGCCTCC (SEQ ID NO: 3); (iii) GTTTGCCGTGTCG (SEQ ID NO: 4); (iv) GTTTGCTGTGTCG (SEQ ID NO: 5); (v) TCCCAAACTCCTG (SEQ ID NO: 6); (vi) TCCCAACCTCCTG (SEQ ID NO: 7); or (vii) any combinations of (i)-(vi).
  • the TS 1 PLXNA4 inhibitory agent comprises the nucleotide sequence selected from: (i) GGTCCTCGCCTCC (SEQ ID NO: 2); (ii)
  • GGTCCTAGCCTCC (SEQ ID NO: 3); (iii) GTTTGCCGTGTCG (SEQ ID NO: 4); (iv) GTTTGCTGTGTCG (SEQ ID NO: 5); (v) TCCCAAACTCCTG (SEQ ID NO: 6); (vi) TCCCAACCTCCTG (SEQ ID NO: 7); or (vii) any combinations of (i)-(vi).
  • the TS 1 PLXNA4 inhibitory agent is an antibody or fragment thereof.
  • antibody and “antibodies” include polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies, single chain Fv antibody fragments, Fab fragments, and F(ab) 2 fragments.
  • the antibody can be a recombinant antibody, humanized antibody, chimeric antibody, modified antibody, monoclonal antibody, polyclonal antibody, miniantibody, dimeric miniantibody, minibody, diabody or tribody or antigen-binding variants, analogues or modified versions thereof.
  • Antibodies having specific binding affinity for PLXNA4 can be produced through standard methods. Alternatively, antibodies may be commercially available, for example, from R&D Systems, Inc., Minneapolis, Minn.
  • antibody refers to intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding and includes chimeric, humanized, fully human, and bispecific antibodies.
  • binding fragments are produced by recombinant DNA techniques.
  • binding fragments are produced by enzymatic or chemical cleavage of intact antibodies.
  • Binding fragments include, but are not limited to, Fab, Fab', F(ab') 2 , Fv, and single-chain antibodies. Unless it is specifically noted, as used herein a "fragment thereof in reference to an antibody refers to an immunespecific fragment, i.e., an antigen-specific or binding fragment.
  • Monoclonal antibodies which are homogeneous populations of antibodies to a particular epitope contained within an antigen, can be prepared using standard hybridoma technology.
  • monoclonal antibodies can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture such as described by Kohler, G. et al, Nature, 1975, 256:495, the human B-cell hybridoma technique (Kosbor et al, Immunology Today, 1983, 4:72; Cole et al, Proc. Natl. Acad. Sci. USA, 1983, 80:2026), and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.
  • Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass thereof.
  • the hybridoma producing the monoclonal antibodies of the invention can be cultivated in vitro or in vivo.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules that are specific for a particular antigen, which are contained in the sera of the immunized animals. Polyclonal antibodies are produced using well-known methods.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human
  • Chimeric antibodies can be produced through standard techniques.
  • Antibody fragments that have specific binding affinity for a component of the complex can be generated by known techniques.
  • fragments include, but are not limited to, F(ab') 2 fragments that can be produced by pepsin digestion of the antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab') 2 fragments.
  • Fab expression libraries can be constructed. See, for example, Huse et al, 1989, Science, 246: 1275.
  • Single chain Fv antibody fragments are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge (e.g., 15 to 18 amino acids), resulting in a single chain polypeptide.
  • Single chain Fv antibody fragments can be produced through standard techniques. See, for example, U.S. Pat. No. 4,946,778.
  • the antibody or antigen-binding fragment thereof is murine. In some embodiments, the antibody or antigen-binding fragment thereof is from mice. In some embodiments, the antibody or antigen-binding fragment thereof is from rat. In other embodiments, the antibody or antigen binding fragment thereof is human. In some
  • the antibody or antigen-binding fragment thereof is recombinant, engineered, humanized and/ or chimeric.
  • treatment means preventing the progression fo the disease, or altering the course of the disorder (for example, but not limited to, slowing the progression of the disorder), or reversing a symptom of the disorder or reducing one or more symptoms and/or one or more biochemical markers in a subject, preventing one or more symptoms from worsening or progressing, promoting recovery or improving prognosis.
  • therapeutic treatment refers to reducing the cognitive deterioration in a subject and/or inhibiting or reducing the level of ⁇ in the brain of a subject that is already inflicted with AD.
  • Measurable lessening includes any statistically significant decline in a measurable marker or symptom, such as measuring ⁇ in the brain by PET scan, or assessing the cognitive improvement with neuropsychological tests such as verbal and perception after treatment.
  • terapéuticaally effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, e.g., a diminishment or prevention of effects associated with various disease states or conditions, such as reduce a symptom of an Alzheimer's disease in the subject.
  • therapeutically effective amount refers to an amount of, for example, a TS 1 PLXNA4 inhibitory agent, as disclosed herein, effective to treat or prevent a disease or disorder in a mammal, preferably a human.
  • a therapeutically effective amount may alleviate one or more symptoms associated with the disease including increasing long-term memory, for example.
  • TS1 PLXNA4 inhibitory agent a TS1 PLXNA4 inhibitory agent
  • a desired site such that desired effect is produced, such as intracranially to brain or specific areas of brain.
  • Stereotactic means can be used to guide intracranial administration if desired.
  • Routes of administration suitable for the methods of the invention include both local and systemic administration. Generally, local administration results in more of the composition being delivered to a specific location as compared to the entire body of the subject, whereas, systemic administration can result in delivery to essentially the entire body of the subject.
  • chemotropic property of NSCs can guide the cells to a specific location with a tissue injury, e.g., brain, even with systemic administration.
  • the agent can be administered by any appropriate route which results in an effective treatment in the subject, including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, and nasal administration.
  • Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion.
  • injection includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub-capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal,
  • systemic administration means the administration of a TS1 PLXNA4 inhibitory agent such that it enters the animal's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • the term "pharmaceutically acceptable carrier” means a
  • composition or vehicle such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • a liquid or solid filler diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • manufacturing aid e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid
  • solvent encapsulating material involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier
  • materials which can serve as pharmaceutically-acceptable carriers include: (i) sugars, such as lactose, glucose and sucrose; (ii) starches, such as corn starch and potato starch; (iii) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, micro crystalline cellulose and cellulose acetate; (iv) powdered tragacanth; (v) malt; (vi) gelatin; (vii) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (viii) excipients, such as cocoa butter and suppository waxes; (ix) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (x) glycols, such as propylene glycol; (xi) polyols, such as glycerin, sorbitol, mannitol
  • the disclosure also provides assays to identify a subject with an increased risk for developing late onset AD.
  • the assay comprises or consists essentially of a system for transforming and identifying at least one nucleic acid polymorphism in a SNP locus described herein in a biological sample of a subject, and a system for computing the likelihood of the subject getting late onset AD on the basis of comparison of the identified nuclei acid at the SNP locus against the AD risk associated alleles described herein.
  • the computing or comparion system which can be a computer implemented system, indicates that at least one of the allele at the SNP locus is identical to the corresponding AD risk associated allele, the subject from which the sample is collected can be diagnosed with increased susceptibility for late onset AD.
  • transformation refers to changing an object or a substance, e.g., biological sample, nucleic acid or protein, into another substance.
  • the transformation can be physical, biological or chemical. Exemplary physical
  • transformation includes, but not limited to, pre-treatment of a biological sample, e.g., from whole blood to blood serum by differential centrifugation.
  • a biological sample e.g., from whole blood to blood serum by differential centrifugation.
  • transformation can involve at least one enzyme and/or a chemical reagent in a reaction.
  • a DNA sample can be digested into fragments by one or more restriction enzyme, or an exogenous molecule can be attached to a fragmented DNA sample with a ligase.
  • a DNA sample can undergo enzymatic replication, e.g., by polymerase chain reaction (PCR).
  • an assay comprising subjecting a test sample from a subject, e.g., a human subject, to at least one genotyping assay that determines the genotypes of at least one (e.g., one, tow, three, four,five, six, seve, eight, nine, ten or more) loci selected from SNP rs277470, rs277472, rs277473, rs277474, rs277476, rs277477, rs277478, rs277479, rs277480, rs277481, rs277483, rs277484, rsl0234979, rs9641933, rsl0225863, rs7799929, rs9656410, rsl 1764790, rs4731860, rsl 1763817, rsl3231950,
  • the loci are selected from lected from: (i) SNP1 , wherein SNP1 is identified by rs277472 on SEQ ID NO: 1 , wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of plexin A4 (PLXNA4); (ii) SNP2, wherein SNP2 is position 132,006,366 of SEQ ID NO: 1 identified by rsl0236235, wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; and (iii) SNP3, wherein SNP3 identified by rsl 1761937 on SEQ ID NO: 1 , wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4.
  • the loci are further selected from: (i) SNP4, wherein SNP4 is identified by rsl593222 on SEQ ID NO: 1 , wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; (ii) SNP5, wherein SNP4 is identified by rs6959579 on SEQ ID NO: 1 , wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; and (iii) SNP6, wherein SNP4 is identified by rsl7166339 on SEQ ID NO: 1 , wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4
  • the assay further comprises selecting a treatment regimen that comprises a therapeutically effective amount of a TS 1 PLXNA4 inhibitory agent when at least one (e.g., one, two, three or more) of the following combinations of SNPs is determined to be present: (i) SNP1 genotype A/A or AJC (or T/T or T/G in the complement) of SEQ ID NO: 1 , wherein SNP1 is identified by rs277472 on SEQ ID NO: 1 , wherein SEQ ID NO.
  • a treatment regimen that comprises a therapeutically effective amount of a TS 1 PLXNA4 inhibitory agent when at least one (e.g., one, two, three or more) of the following combinations of SNPs is determined to be present: (i) SNP1 genotype A/A or AJC (or T/T or T/G in the complement) of SEQ ID NO: 1 , wherein SNP1 is identified by rs277472 on SEQ ID NO: 1 , wherein SEQ ID
  • SNP2 genotype T/T or T/C (or A/A or AJC in the complement) of SEQ ID NO: 1 , wherein SNP2 is position 132,006,366 of SEQ ID NO: 1 identified by rsl0236235, wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; and (iii) SNP3 genotype C/C or C/A (or G/G or G/T in the complement) of SEQ ID NO: 1 , wherein SNP3 identified by rsl 1761937 on SEQ ID NO: 1 , wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4.
  • the disclosure provides a method for treating a subject, e.g. a human subject having or at risk for Alzeimer's disease.
  • the method comprising
  • a TS 1 PLXNA4 inhibitory agent to the subjet which is determined to carry at least one (e.g., one, two, threeor more) SNPs selected from: (i) SNP1 genotype A/A or AJC (or T/T or T/G in the complement) of SEQ ID NO: 1 , wherein SNPl is identified by rs277472 on SEQ ID NO: 1 , wherein SEQ ID NO.
  • SNP2 genotype T/T or T/C (or A/A or AJC in the complement) of SEQ ID NO: 1 , wherein SNP2 is position 132,006,366 of SEQ ID NO: 1 identified by rsl0236235, wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; and (iii) SNP3 genotype C/C or C/A (or G/G or G/T in the complement) of SEQ ID NO: 1 , wherein SNP3 identified by rsl 1761937 on SEQ ID NO: 1 , wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4.
  • the disclosure provides a method for selecting a subject having or at risk for AD, wherein the subject is susceptible to treatment with a TS l PLXNA4 inhibitory agent.
  • the method comprising: (a) contacting a biological sample with at least one (e.g., one, two, three, four or more) oligonucleotide capable of interrogating whether or not the biological sample comprises one or more of the single nucleotide polymorphisms (SNPs) selected from: (i) SNPl genotype A/A or AJC (or T/T or T/G in the complement) of SEQ ID NO: 1 , wherein SNPl is identified by rs277472 on SEQ ID NO: 1 , wherein SEQ ID NO.
  • SNPs single nucleotide polymorphisms
  • SNP1 is a portion of genomic nucleic acid sequence of plexin A4 (PLXNA4); (ii) SNP2 genotype T/T or T/C (or A/A or AJC in the complement) of SEQ ID NO: 1 , wherein SNP2 is position 132,006,366 of SEQ ID NO: 1 identified by rsl0236235, wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; and (iii) SNP3 genotype C/C or C/A (or G/G or G/T in the complement) of SEQ ID NO: 1 , wherein SNP3 identified by rsl 1761937 on SEQ ID NO: 1 , wherein the SEQ ID NO.
  • PLXNA4 plexin A4
  • 1 is a portion of genomic nucleic acid sequence of PLXNA4; and (b) identifying Alzheimer's disease in the subject as susceptible for treatment with a TSl PLXNA4 inhibitory agent when at least one (e.g., one, two, or three) of the SNPs of (i)-(iii) is detected in the biological sample, and identifying Alzheimer's disease in the subject as poorly or non-responsive to treatment with the TS l PLXNA4 inhibitory agent when none of the SNPs of (i)-(iii) is detected in the biological sample.
  • a TSl PLXNA4 inhibitory agent when at least one (e.g., one, two, or three) of the SNPs of (i)-(iii) is detected in the biological sample, and identifying Alzheimer's disease in the subject as poorly or non-responsive to treatment with the TS l PLXNA4 inhibitory agent when none of the SNPs of (i)-(iii) is detected in the biological sample
  • the disclosure provide an assay comprising: (a) contacting a biological sample obtained from a subject with a detectable antibody specific for PLXNA4 or detectable nucleic acid complementary to at least part of PLXNA4, e.g., SNP rs277472, SNP rsl0236235, and/or SNP rsl 17619371ocus; (b) washing the sample to remove unbound antibody or unbound nucleic acid; (c) measuring the intensity of the signal from the bound, detectable antibody or bound detectable nucleic acid; (d) comparing the measured intensity of the signal with a reference value and if the measured intensity is normal and/or increased relative to the reference value; the subject is identified as having or at risk for AD.
  • a detectable antibody specific for PLXNA4 or detectable nucleic acid complementary to at least part of PLXNA4 e.g., SNP rs277472, SNP rsl0236235, and/or SNP rsl 1761
  • Another aspect of the present invention relates to a system for obtaining data from at least one test sample obtained from at least one subject, the system comprising: (a) a determination module configured to receive said at least one test sample and perform at least one analysis on said at least one test sample to determine the presence or absence of at least one of the following conditions: (i) the level of expression or amount of PLXNA4 (e.g., PLXNA4 isoform TS l or TS3) is higher than a pre-determined level; (ii) at least one copy of a single nucleotide polymorphism (SNP) selected from: (1) SNP1 genotype A/A or AJC (or T/T or T/G in the complement) of SEQ ID NO: 1 , wherein SNP1 is identified by rs277472 on SEQ ID NO: 1 , wherein SEQ ID NO.
  • SNP single nucleotide polymorphism
  • SNP1 is a portion of genomic nucleic acid sequence of plexin A4 (PLXNA4); (2) SNP2 genotype T/T or T/C (or A/A or A/C in the complement) of SEQ ID NO: 1 , wherein SNP2 is position 132,006,366 of SEQ ID NO: 1 identified by rs 10236235, wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; (3) SNP3 genotype C/C or C/A (or G/G or G/T in the complement) of SEQ ID NO: 1 , wherein SNP3 identified by rsl 1761937 on SEQ ID NO: 1 , wherein the SEQ ID NO.
  • PLXNA4 plexin A4
  • 1 is a portion of genomic nucleic acid sequence of PLXNA4; and (4) any combinations thereof; (b) a storage device configured to store data output from said determination module; and (c) a display module for displaying a content based in part on the data output from said determination module, wherein the content comprises a signal indicative of the presence of at least one of these conditions determined by the determination module, or a signal indicative of the absence of at least one of these conditions determined by the determination module.
  • the content displayed from the display module of the system as disclosed herein can further comprise a signal indicative of the subject being recommended to receive a particular treatment regimen, for example, if the subject has one or more of the above conditions, a signal is produced to recommend the subject be administered an AD therapy, for example, but not limited to, a TS l PLXNA4 inhibitory agent.
  • the subject is recommended for AD therapy, e.g., a treatment with a composition comprising a TS l PLXNA4 inhibitory agent, where the content from the display module produces a signal indicative of at least one of: (a) increased expression level or amount of TS l or TS3 PLXNA4 isoform; and (b) presence of at least one AD risk associated SNP as disclosed herein.
  • AD therapy e.g., a treatment with a composition comprising a TS l PLXNA4 inhibitory agent, where the content from the display module produces a signal indicative of at least one of: (a) increased expression level or amount of TS l or TS3 PLXNA4 isoform; and (b) presence of at least one AD risk associated SNP as disclosed herein.
  • a subject is not recommended for AD therapy, e.g., a treatment with a composition comprising a TS l PLXNA4 inhibitory agent, where the content from the display module produces a signal indicative of at least one of: (a) lower or reduced expression level or amount of TS1 or TS3 PLXNA4 isoform; and (b) absence of at least one AD risk associated SNP as disclosed herein.
  • the disclosure provides a method of determining if a subject is responsive to a TS1 PLXNA4 inhibitory agent.
  • the method comprising assaying a blood sample for the presence of at least one (e.g., one, two, or three) of: (i) SNPl genotype A/A or A/C (or T/T or T/G in the complement) of SEQ ID NO: 1, wherein SNPl is identified by rs277472 on SEQ ID NO: 1, wherein SEQ ID NO.
  • SNP2 genotype T/T or T/C or A/A or A/C in the complement
  • SNP2 is position 132,006,366 of SEQ ID NO: 1 identified by rs 10236235, wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4
  • SNP3 genotype C/C or C/A or G/G or G/T in the complement
  • SNP3 identified by rsl 1761937 on SEQ ID NO: 1 wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4.
  • SNPs are single base positions in DNA at which different alleles, or alternative nucleotides, exist in a population.
  • the SNP position (interchangeably referred to herein as SNP, SNP site, SNP allele or SNP locus) is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of the populations).
  • An individual can be homozygous or heterozygous for an allele at each SNP position.
  • a SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP is an amino acid coding sequence.
  • a SNP can arise from a substitution of one nucleotide for another at the polymorphic site. Substitutions can be transitions or transversions. A transition is the replacement of one purine nucleotide by another purine nucleotide, or one pyrimidine by another pyrimidine. A transversion is the replacement of a purine by a pyrimidine, or vice versa.
  • a SNP can also be a single base insertion or deletion variant referred to as an "in/del” (Weber et al., "Human diallelic insertion/deletion polymorphisms", Am J Hum Genet October 2002; 71(4):854-62).
  • a synonymous codon change, or silent mutation/SNP is one that does not result in a change of amino acid due to the degeneracy of the genetic code.
  • a substitution that changes a codon coding for one amino acid to a codon coding for a different amino acid is referred to as a missense mutation.
  • a nonsense mutation results in a type of non-synonymous codon change in which a stop codon is formed, thereby leading to premature termination of a polypeptide chain and a truncated protein.
  • a read-through mutation is another type of non-synonymous codon change that causes the destruction of a stop codon, thereby resulting in an extended polypeptide product.
  • SNPs can be bi-, tri-, or tetra-allelic, the vast majority of the SNPs are bi-allelic, and are thus often referred to as "bi-allelic markers", or "di-allelic markers”.
  • a major database of human SNPs is maintained at NCBI as dbSNP, and it contains data for unique human SNPs consisting of 1.1 x 10 8 submitted SNP (identified by an "ss" number) and 2.4 x 10 7 reference SNP (identified by an "rs” number), as of Build Hisotry 131 : human_9606 based on GRCh37 available from the NCBI website.
  • the rs numbers are unique, do not change and allow analysis of the particularly identified SNP in any genetic sample.
  • the SNPs described herein are identified by an "rs" number.
  • One of skill in the art will be able to determine the position of a specific SNP within a respective chromosome.
  • SNP Single-Nucleotide Polymorphism Incidence in Human and Orangutan Xq: Deserts and Recent Coalescences. Genomics 71 : 78-88. ).
  • An association study of a SNP and a specific disorder involves determining the presence or frequency of the SNP allele in biological samples from subjects with the disorder of interest, such as Alzheimer's disease, and comparing the information to that of controls (i.e., individuals who do not have the disorder; controls can be also referred to as "healthy” or "normal” individuals) who are preferably of similar age and race.
  • controls i.e., individuals who do not have the disorder; controls can be also referred to as "healthy” or "normal” individuals
  • the appropriate selection of patients and controls is important to the success of SNP association studies. Therefore, a pool of individuals with well-characterized phenotypes is desirable.
  • Association studies can be conducted within the general population and are not limited to studies performed on related individuals in affected families (linkage studies).
  • a SNP can be screened in any biological sample obtained from an individual or a subject diagnosed with or at risk of a disease or disorder, e.g., Alzheimer's disease. If an allele herein discovered as an AD risk allele is identified, the subject can be identified as at greater risk of developing AD than a subject who is not carrying that alleles.
  • a disease or disorder e.g., Alzheimer's disease.
  • SNP alleles sometimes referred to as polymorphisms or polymorphic alleles
  • the AD is late-onset form. Mutations or alleles identifying a subject with an increased risk of developing a disorder, for example, late onset AD, are also referred to as "susceptibility" alleles, or mutations.
  • nucleic acid molecules can be double-stranded molecules and that reference to a particular site on one strand refers, as well, to the corresponding site on a complementary strand.
  • reference to an adenine, a thymine (uridine), a cytosine, or a guanine at a particular site on one strand of a nucleic acid molecule also defines the thymine (uridine), adenine, guanine, or cytosine (respectively) at the corresponding site on a complementary strand of the nucleic acid molecule.
  • Probes and primers can be designed to hybridize to either strand and SNP genotyping methods disclosed herein can generally target either strand. Accordingly, the claims are intended to cover analysis of the opposite strand as well. For the opposite-strand analysis.
  • Identification method of SNPs can be of either a positive-type (inclusion of an allele) or a negative- type (exclusion of an allele).
  • Positive-type methods determine the identity of a nucleotide contained in a polymorphic site
  • negative-type methods determine the identity of a nucleotide not present in a polymorphic site.
  • a wild-type site can be identified either as wild-type or not mutant.
  • a site can be positively determined to be either adenine or cytosine or negatively determined to be not adenine (and thus cytosine) or not cytosine (and thus adenine).
  • the nucleic acid sequences of the gene's allelic variants, or portions thereof can be the basis for probes or primers, e.g., in methods for determining the identity of the allelic variant of the polymorphic region.
  • nucleic acid probes or primers can be used in the methods of the present invention to determine whether a subject is at risk of developing disease such as Alzheimer's disease.
  • One of skill in the art can readily access the nucleic acid sequences spanning the SNPs described herein through the NCBI dbSNP database with the "rs" number uniquely assigned to each SNPs described herein.
  • a skilled artisan can readily design and optimize primers or probes based on the flanking sequences of the SNP loci described herein.
  • polymorphisms of PLXNA4 dislcosed herein can be detected directly or indirectly using any of a variety of suitable methods including fluorescent polarization, mass spectroscopy, and the like.
  • suitable methods comprise direct or indirect sequencing methods, restriction site analysis, hybridization methods, nucleic acid amplification methods, gel migration methods, the use of antibodies that are specific for the proteins encoded by the different alleles of the polymorphism, or by other suitable means.
  • many such methods are well known in the art and are described, for example in T. Maniatis et al., Molecular Cloning, a Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, New York (1989), J.W.
  • Methods to measure gene expression products associated with AD risk associated SNPs described herein are well known to a skilled artisan. Such methods to measure gene expression products, e.g., protein level, include ELISA (enzyme linked immunosorbent assay), western blot, immunoprecipitation, immunofluorescence using detection reagents such as an antibody or protein binding agents. Alternatively, a peptide can be detected in a subject by introducing into a subject a labeled anti-peptide antibody and other types of detection agent.
  • the antibody can be labeled with a radioactive marker whose presence and location in the subject is detected by standard imaging techniques, particularly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample.
  • any approach that detects mutations or polymorphisms in a gene can be used, including but not limited to single-strand conformational polymorphism (SSCP) analysis (Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766-2770), heteroduplex analysis (Prior et al. (1995) Hum. Mutat. 5:263-268), oligonucleotide ligation (Nickerson et al. (1990) Proc. Natl. Acad. Sci. USA 87:8923-8927) and hybridization assays (Conner et al. (1983) Proc. Natl. Acad. Sci. USA 80:278-282).
  • SSCP single-strand conformational polymorphism
  • the gene expression products as described herein can be determined by determining the level of messenger RNA (mRNA) expression of genes associated with SNPs described herein (e.g., PLXNA 4).
  • mRNA messenger RNA
  • Such molecules can be isolated, derived, or amplified from a biological sample, such as body fluids. Detection of mRNA expression is known by persons skilled in the art, and comprise, for example but not limited to, PCR procedures, RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization methods etc.
  • Nucleic acid or ribonucleic acid (RNA) molecules can be isolated from a particular biological sample using any of a number of procedures, which are well-known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample. For example, freeze-thaw and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from solid materials; heat and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from urine; and proteinase K extraction can be used to obtain nucleic acid from blood (Roiff, A et al. PCR: Clinical Diagnostics and
  • the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes within a nucleic acid sample or library, (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a DNA polymerase, and (iii) screening the PCR products for a band of the correct size.
  • the primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to each strand of the genomic locus to be amplified.
  • mRNA level of gene expression products described herein can be determined by reverse-transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) or real-time PCR methods. Methods of RT-PCR and QRT-PCR are well known in the art.
  • an allelic discrimination method can be used for identifying the genotype of SNPs described herein.
  • the allelic discrimination method involves use of a first oligonucleotide probe which anneals with a target portion of the individual's genome. Because the nucleotide residue at this position differs, the first probe is completely complementary to only one of the two alleles.
  • allelic discrimination method also involves use of at least one, and preferably a pair of amplification primers for amplifying a reference region, for example, at least a portion of the flanking region including the SNP locus of interest.
  • the probe in some embodiments is a DNA oligonucleotide having a length in the range from about 20 to about 40 nucleotide residues, preferably from about 20 to about 30 nucleotide residues, and more preferably having a length of about 25 nucleotide residues.
  • the probe is rendered incapable of extension by a PCR-catalyzing enzyme such as Taq polymerase, for example by having a fluorescent probe attached at one or both ends thereof.
  • a PCR-catalyzing enzyme such as Taq polymerase
  • the probes are preferably detectably labeled.
  • Exemplary labels include radionuclides, light-absorbing chemical moieties (e.g.
  • the label is a fluorescent moiety, such as 6-carboxyfluorescein (FAM), 6-carboxy-4,7,2',7'-tetrachlorofluoroscein (TET), rhodamine, JOE (2,7-dimethoxy- 4,5-dichloro-6-carboxyfluorescein), HEX (hexachloro-6-carboxyfluorescein), or VIC.
  • FAM 6-carboxyfluorescein
  • TET 6-carboxy-4,7,2',7'-tetrachlorofluoroscein
  • rhodamine rhodamine
  • JOE 2,7-dimethoxy- 4,5-dichloro-6-carboxyfluorescein
  • HEX hexachloro-6-carboxyfluorescein
  • the probe can comprise both a fluorescent label and a fluorescence-quenching moiety such as 6-carboxy-N,N,N',N'-tetramethylrhodamine
  • TAMRA 4-(4'-dimethlyaminophenylazo)benzoic acid
  • DBCYL 4-(4'-dimethlyaminophenylazo)benzoic acid
  • the fluorescent intensity of the fluorescent label is diminished.
  • the intensity of the fluorescent label is no longer diminished.
  • the probe of the present invention has a fluorescent label attached at or near (i.e. within about 10 nucleotide residues of) one end of the probe and a fluorescence-quenching moiety attached at or near the other end.
  • Degradation of the probe by a PCR-catalyzing enzyme releases at least one of the fluorescent label and the fluorescence-quenching moiety from the probe, thereby discontinuing fluorescence quenching and increasing the detectable intensity of the fluorescent labels.
  • cleavage of the probe (which, as discussed above, is correlated with complete complementarity of the probe with the target portion) can be detected as an increase in fluorescence of the assay mixture.
  • the assay mixture can contain a first probe which is completely complementary to the target portion of a first AD associated SNP and to which a first label is attached, and a second probe which is completely complementary to the target portion of a second AD risk associated SNP.
  • the probes are detectably different from each other, having, for example, detectably different size, absorbance, excitation, or emission spectra, radiative emission properties, or the like.
  • a first probe can be completely complementary to the target portion of the polymorphism and have FAM and TAMRA attached at or near opposite ends thereof.
  • the first probe can be used in the method of the present invention together with a second probe which is completely complementary to the target portion of another AD risk associated and has TET and TAMRA attached at or near opposite ends thereof.
  • Fluorescent enhancement of FAM i.e. effected by cessation of fluorescence quenching upon degradation of the first probe by Taq polymerase
  • one wavelength e.g. 518
  • TET fluorescent enhancement of TET
  • a different wavelength e.g. 582 nanometers.
  • more than one SNP described herein can be detected, providing a better diagnosis and more reliable prediction of AD susceptibility in a subject.
  • the probe comprises a nucleotide sequence substantially complementary to: (i) GGTCCTCGCCTCC (SEQ ID NO: 2); (ii) GGTCCTAGCCTCC (SEQ ID NO: 3); (iii) GTTTGCCGTGTCG (SEQ ID NO: 4); (iv) GTTTGCTGTGTCG (SEQ ID NO: 5); (v) TCCCAAACTCCTG (SEQ ID NO: 6); (vi) TCCCAACCTCCTG (SEQ ID NO: 7); or (vii) any combinations of (i)-(vi).
  • the probe comprises the nucleotide sequence selected from: (i) GGTCCTCGCCTCC (SEQ ID NO: 2); (ii) GGTCCTAGCCTCC (SEQ ID NO: 3); (iii) GTTTGCCGTGTCG (SEQ ID NO: 4); (iv) GTTTGCTGTGTCG (SEQ ID NO: 5); (v) TCCCAAACTCCTG (SEQ ID NO: 6); (vi) TCCCAACCTCCTG (SEQ ID NO: 7); or (vii) any combinations of (i)-(vi).
  • allelic discrimination method suitable for use in detection of SNPs employs "molecular beacons". Detailed description of this methodology can be found in Kostrikis et al., Science 1998, 279: 1228-1229, content of which is incorporated herein by reference in its entirety.
  • the molecular beacon probe comprises a nucleotide sequence substantially complementary to: (i) GGTCCTCGCCTCC (SEQ ID NO: 2); (ii) GGTCCTAGCCTCC (SEQ ID NO: 3); (iii) GTTTGCCGTGTCG (SEQ ID NO: 4); (iv) GTTTGCTGTGTCG (SEQ ID NO: 5); (v) TCCCAAACTCCTG (SEQ ID NO: 6); (vi) TCCCAACCTCCTG (SEQ ID NO: 7); or (vii) any combinations of (i)-(vi).
  • the molecular beacon probe comprises the nucleotide sequence selected from: (i) GGTCCTCGCCTCC (SEQ ID NO: 2); (ii) GGTCCTAGCCTCC (SEQ ID NO: 3); (iii) GTTTGCCGTGTCG (SEQ ID NO: 4); (iv) GTTTGCTGTGTCG (SEQ ID NO: 5); (v) TCCCAAACTCCTG (SEQ ID NO: 6); (vi) TCCCAACCTCCTG (SEQ ID NO: 7); or (vii) any combinations of (i)-(vi).
  • microarrays comprising a multiplicity of sequences is becoming increasingly common in the art. Accordingly, a microarray having at least one
  • oligonucleotide probe as described above, appended thereon, can be used for SNP genotyping.
  • restriction enzymes can be utilized to identify variances or a polymorphic site using "restriction fragment length polymorphism" (RFLP) analysis (Lentes et al. , Nucleic Acids Res. 16:2359 (1988); and C.K. McQuitty et al, Hum. Genet. 93:225 (1994)).
  • RFLP restriction fragment length polymorphism
  • at least one target polynucleotide is digested with at least one restriction enzyme and the resulting restriction fragments are separated based on mobility in a gel. Typically, smaller fragments migrate faster than larger fragments. Consequently, a target polynucleotide that contains a particular restriction enzyme recognition site will be digested into two or more smaller fragments, which will migrate faster than a larger fragment lacking the restriction enzyme site.
  • Knowledge of the nucleotide sequence of the target will be digested into two or more smaller fragments, which will migrate faster than a larger fragment lacking the restriction enzyme site.
  • restriction site analysis of particular nucleotide sequence to identify a nucleotide at a polymorphic site is determined by the presence or absence of a restriction enzyme site.
  • restriction enzymes A large number of restriction enzymes are known in the art and, taken together, they are capable of recognizing at least one allele of many polymorphisms.
  • SNPs single nucleotide polymorphisms
  • SNPs single nucleotide polymorphisms
  • a number of approaches use DNA ligase, an enzyme that can join two adjacent oligonucleotides hybridized to a DNA template.
  • OLA Oligonucleotide ligaton assay
  • ASO allele-specific oligonucleotides
  • Numerous approaches cane be used for the detection of the ligated products, for example the ASOs with differentially labeled with fluorescent of hapten labels and ligated products detected by fluorogenic of colorimetric enzyme-linked immunosorbent assays (Tobe et al, Nuclic Acid Res, 1996;24;3728-32).
  • a morbidity modifier taqgs or variation in probe length coupled with floursecence detection enables the multiplex genotyping of several single nucleotide substitutions in a single tube (Baron et al, 1997; Clinical Chem., 43, 1984-6).
  • ASOs can be spotted at specific locations or addresses on a chip, PCR amplified DNA can then be added and ligation to labeled oligonucleotides at specific addreses on the array measured (Zhong et al, Proc Natl Acad Sci 2003; 100, 11559-64).
  • Single base-extension or minisequencing involves annealing an oligonucleotide primer to the single strand of a PCR product and the addition of a single dideoxynucleotide by thermal DNA polymerase.
  • the oligonucleotide is designed to be one base short of the mutation site.
  • the dideoxynucleotide incorporated is complementary to the base at the mutation site.
  • Approaches cans uses different fluorescent tags or haptens for each of the four different dideoxynucleotides (Pastinen et al, Clin Chem 1996, 42, 1391-7). The
  • matrix-assisted laser adsorption/ionization time -of flight mass spectrometry or MALDI-TOF (Li et al, Electrophorosis, 1999,20; 1258- 65), which is quantitative and can be used to calculate the relative allele abundance making the approach suitable for other applications such as gene dosage studies (for example for estimation of allele frequencies on pooled DNA samples).
  • Minisequencing or Microsequencing by MALDI-TOF can be performed by means known by persons skilled in the art.
  • some embodiments can use the Sequenom's Mass Array Technology (www.sequenom.com) (Sauser et al, Nucleic Acid Res, 2000, 28;E13 and Sauser et al, Nucleic Acid Res 2000, 28: E100). and also the GOOD Assay (Sauer S et al, Nucleic Acid Res, 2000; 28, E13 and Sauer et al, Nucleic Acid Res, 2000;28:E100).
  • variations of MALDI-TOF can be performed for analysis of variances in the genes associated with SNPs described herein.
  • MALDI and electrospray ioinization ESI
  • ESI electrospray ioinization
  • Allele-specific Amplification is also known as amplification refectory mutation system (ARMS) uses allele specific oligonucleotides (ASO) PCR primers and is an well established and known PCR based method for genotyping (Newton et al, J Med Genet, 1991;28;248-51). Typically, one of the two oligonucleotide primers used for the PCR binds to the mutation site, and amplification only takes place if the nucleotide of the mutation is present, with a mismatch being refractory to amplification. The resulting PCR Products can be analyzed by any means known to persons skilled in the art.
  • MS-PCR mutagenically separated PCR
  • Normal or mutant alleles can be genotyped by measuring the binding of allele- specific oligonucleotides (ASO) hybridization probes.
  • ASO allele-specific oligonucleotides
  • two ASO probes, one complementary to the normal allele and the other to the mutant allele are hybridized to PCR-amp lifted DNA spanning the mutation site.
  • the amplified products can be immobilized on a solid surface and hybridization to radiolabeled oligonucleotides such as known as a 'dot-blot' assay.
  • the binding of the PCR products containing a quantifiable label eg biotin or fluorescent labels
  • telomere binding of PCR products containing a quantifiable lable for example but not limited to biotin or fluorescent labels
  • a solid phase allele-specific oligonucleotide can be measured.
  • the use of microarrays comprising hundreds of ASO immobilized onto a solid support surfaces to form an array of ASO can also be used for large scale genotyping of multiple single quantifiable lable (for example but not limited to biotin or fluorescent labels)
  • polymorphisms simultaneously, for example Affymetrix GENECHIP® Mapping 10K Array, which can easily be performed by persons skilled in the art.
  • the ASO comprises a nucleotide sequence substantially complementary to: (i) GGTCCTCGCCTCC (SEQ ID NO: 2); (ii) GGTCCTAGCCTCC (SEQ ID NO: 3); (iii) GTTTGCCGTGTCG (SEQ ID NO: 4); (iv) GTTTGCTGTGTCG (SEQ ID NO: 5); (v) TCCCAAACTCCTG (SEQ ID NO: 6); (vi) TCCCAACCTCCTG (SEQ ID NO: 7); or (vii) any combinations of (i)-(vi).
  • the ASO comprises a nucleotide sequence selected from (i) GGTCCTCGCCTCC (SEQ ID NO: 2); (ii) GGTCCTAGCCTCC (SEQ ID NO: 3); (iii) GTTTGCCGTGTCG (SEQ ID NO: 4); (iv) GTTTGCTGTGTCG (SEQ ID NO: 5); (v) TCCCAAACTCCTG (SEQ ID NO: 6); (vi) TCCCAACCTCCTG (SEQ ID NO: 7); or (vii) any combinations of (i)-(vi).
  • Homogenous assays also called “closed tube” arrays, genomic DNA and all the reagents required for the amplification and genotyping are added simultaneously. Genotyping can be achieved without any post-amplification processing.
  • one such homogenous assay is the 5'flurogenic nuclease assay, also known as the TAQMAN® Assay (Livak et al, Genet Anal, 1999; 14: 143-9) and in alternative embodiments Melting curve analyses of FRET probes are used.
  • TAQMAN® Assay Melting curve analyses of FRET probes are used.
  • Such methods are carried out using "real-time" theromcyclers, and utilize two dual-labeled ASO hybridization probes complementary to normal and mutant alleles, where the two probes have different reported labels but a common quencher dye.
  • the changes in fluorescence characteristics of the probes upon binding to PCR products of target genes during amplification enables "realtime” monitoring of PCR amplification and differences in affinity of the fluorogenic probes for the PCR products of normal and mutant genes enables differentiation of genotypes.
  • the approach uses two dual-labeled ASO hybridization probes complementary to the mutant and normal alleles. The two probes have different fluorescent reported dyes but a common quencher dye. When intact, the probes do not fluoresces due to the proximity of the reporter and quencher dyes.
  • two probes compete for hybridization to their target sequences, downstream of the primer sites and are subsequently cleaved by 5' nuclease activity of Thermophilis aquaticus (Taq) polymerase as the primer is extended, resulting in the separation of the reporter dyes from the quencher.
  • Genotyping is determined by measurement of the fluorescent intensity of the two reporter dyes after PCR amplification.
  • the reaction includes two oligonucleotide probes which when in close proximity forms a fluorescent complex, where one probe often termed the “mutant sensor” probe is designed to specifically hybridize across the mutation site and the other probe (often referred to as the “anchor probe") hybridizes to an adjacent site.
  • Fluorescent light is emitted by the "donor” excites the “acceptor” fluorphore creasing a unique fluorogenic complex, which only forms when the probes bind to adjacent sites on the amplified DNA.
  • the "sensor” probe is complementary to either the normal or the mutant allele.
  • LCGREENTM is a sensitive highly fluorogenic double-stranded DNA (dsDNA) binding dye that is used to detect the dissociation of unlabelled probes (Liew et al, Clin Chem, 2004; 50; 1156-64 and Zhou et al, Clin Chem, 2005; 51;1761-2).
  • the method uses unlabeled allele-specific oligonucleotides probes that are perfectly complementary either to the mutant or normal allele, and the mismatch of the ASO/template double strand DNA complex results in a lower melting temperature and an earlier reduction in fluorescent signal form the dsDNA binding dye with increasing temperature.
  • the OLA can also be used for FRET Probes (Chen et al, 1998; 8:549-56), for example, the PCR/ligation mixture can contain PCR primers, DNA polymerase without 5 ' nuclease activity, thermal stable DNA ligase and oligonucleotides for the ligation reaction.
  • the ligation of the allele-specific oligonucleotides have a different acceptor fluorophore and the third ligation oligonucleotide, which binds adjacently to the ASO has a donor fluorophore, and the three ligation oligonucleotides are designed to have a lower melting temperature for the PCR primers to prevent their interference in the PCR amplification. Following PCR, the temperature is lowered to allow ligation to proceed, which results in FRET between the donor and acceptor dyes, and alleles can be disconcerted by comparing the fluorescence emission of the two dyes.
  • Alternatives to homogenous PCR- and hybridization -based techniques are also encompassed. For example, molecular beacons (Tyagi et al, Nat Biotech, 1998, 16: 49-53) and SCORPION® probes (Thelwell et al, Nucleic Acid Res, 2000; 28;3752-610).
  • the OLA can also be performed by the use of FRET probes (Chen et al, Genome Res, 1998;8: 549-56).
  • the PCR/ligation mix contains PCR primers, a thermostable DNA polymerase without 5 ' exonuclease activity (to prevent the cleavage of ligation probes during the ligation phase), a thermostable DNA ligase as well as the oligonucleotides for the ligation reaction.
  • the ligation of the ASO each have a different acceptor flurophore and the third ligation oligonucleotide which binds adjacently to the ASO has a donor flurophore.
  • the three ligation oligonucleotides are designed to horrin a lower melting temperature than the annealing temperature for the PCR primers to prevent their interference in PCR amplification. Following PCR, the temperature is lowered to allow ligation to proceed. Ligation results in FRET between donor and acceptor dyes, and alleles can be discerned by comparing the fluorescence emission of the two dyes.
  • Molecular Beacons (Tyagi et al, Nat Biotech 1998; 16; 49-53) and SCORPION® Probes (Thelwell et al, Nucleic Acid Res 2000;28;3752-61).
  • Molecular Beacons are comprised of oligonucleotides that have fluorescent reporter and dyes at their 5' and 3' ends, with the central portion of the oligonucleotide hybridizing across the target sequence, but the 5' and 3' flanking regions are complementary to each other.
  • polymorphisms can be detected by genotyping using a homogenous or real-time analysis on whole blood samples, without the need for DNA extraction or real-time PCR.
  • a homogenous or real-time analysis on whole blood samples, without the need for DNA extraction or real-time PCR.
  • Such a method is compatible with FRET and TAQMAN® (Castley et al, Clin Chem, 2005;51; 2025-30) enabling extremely rapid screening for the particular polymorphism of interest.
  • FP Fluorescent Polarization
  • the degree to which the emitted light remains polarized in a particular plane is proportional to the speed at which the molecules rotate and tumble in solution.
  • FP is directly related to the molecular weight of a fluorescent species. Therefore, when a small fluorescent molecule is incorporated into a larger molecule, there is an increase in FP.
  • FP can be used in for genotyping of polymorphisms of interest (Chen et al, Genome Res, 1999; 9: 492-8 and Latif et al, Genome Res, 2001; 11;436-40).
  • FP can be utilized in 5' nuclease assay (as described above), where the oligonucleotide probe is digested to a lower molecule weight species, for example is amenable to analysis by FP, but with the added benefit of not requiring a quencher.
  • Perlkin-Elmers AcycloPrimeTM-FP SNP Detection Kit can be used as a FP minisequencing method.
  • unicoportated primers and nucleotides are degraded enzymatially, the enzymes heat inactivated and a miniseqencing reaction using DNA polymerase and fluorescent-labelled dideoxynucleotides performed.
  • FP is then measured, typically in a 96- to 386-well plate format on a FP-plate reader.
  • the primer extension reaction and analysis is performed using PYROSEQUENCINGTM (Uppsala, Sweden) which essentially is sequencing by synthesis.
  • a sequencing primer designed directly next to the nucleic acid differing between the disease-causing mutation and the normal allele or the different SNP alleles is first hybridized to a single stranded, PCR amplified DNA template from the individual, and incubated with the enzymes, DNA polymerase, ATP sulfurylase, luciferase and apyrase, and the substrates, adenosine 5 ' phosphosulfate (APS) and luciferin.
  • APS adenosine 5 ' phosphosulfate
  • deoxynucleotide triphosphates dNTP
  • DNA polymerase catalyzes the incorporation of the dNTP into the standard DNA strand.
  • PPi pyrophosphate
  • ATP sulfurylase converts PPi to ATP in the presence of adenosine 5 ' phosphosulfate.
  • This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP.
  • the light produced in the luciferase-catalyzed reaction is detected by a charge coupled device (CCD) camera and seen as a peak in a PYROGRAMTM.
  • CCD charge coupled device
  • Each light signal is proportional to the number of nucleotides incorporated and allows a clear determination of the presence or absence of, for example, the mutation or polymorphism.
  • apyrase a nucleotide degrading enzyme, continuously degrades unincorporated dNTPs and excess ATP.
  • another dNTP is added which corresponds to the dNTP present in for example the selected SNP. Addition of dNTPs is performed one at a time.
  • Deoxyadenosine alfa-thio triphosphate dATPS
  • dATP deoxyadenosine triphosphate
  • a long-range PCR (LR-PCR) is used to detect mutations or polymorphisms of the present invention.
  • LR-PCR products are genotyped for mutations or polymorphisms using any genotyping methods known to one skilled in the art, and
  • Molecular beacons also contain fluorescent and quenching dyes, but FRET only occurs when the quenching dye is directly adjacent to the fluorescent dye.
  • Molecular beacons are designed to adopt a hairpin structure while free in solution, bringing the fluorescent dye and quencher in close proximity. Therefore, for example, two different molecular beacons are designed, one recognizing the mutation or polymorphism and the other the corresponding wildtype allele. When the molecular beacons hybridize to the nucleic acids, the fluorescent dye and quencher are separated, FRET does not occur, and the fluorescent dye emits light upon irradiation. Unlike TaqMan probes, molecular beacons are designed to remain intact during the amplification reaction, and must rebind to target in every cycle for signal measurement.
  • TaqMan probes and molecular beacons allow multiple DNA species to be measured in the same sample (multiplex PCR), since fluorescent dyes with different emission spectra can be attached to the different probes, e.g. different dyes are used in making the probes for different disease-causing and SNP alleles.
  • Multiplex PCR also allows internal controls to be co-amplified and permits allele discrimination in single-tube assays. (Ambion Inc, Austin, TX, TechNotes 8(1) - February 2001, Real-time PCR goes prime time).
  • Another method to detect mutations or polymorphisms is by using fluorescence tagged dNTP/ddNTPs.
  • a standard nucleic acid sequencing gel can be used to detect the fluorescent label incorporated into the PCR amplification product.
  • a sequencing primer is designed to anneal next to the base differentiating the disease-causing and normal allele or the selected SNP alleles.
  • a primer extension reaction is performed using chain terminating dideoxyribonucleoside triphosphates (ddNTPs) labeled with a fluorescent dye, one label attached to the ddNTP to be added to the standard nucleic acid and another to the ddNTP to be added to the target nucleic acid.
  • ddNTPs dideoxyribonucleoside triphosphates
  • an INVADER® assay can be used (Third Wave Technologies, Inc (Madison, WI)). This assay is generally based upon a structure-specific nuclease activity of a variety of enzymes, which are used to cleave a target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof in a sample (see, e.g. U.S. Patent No. 6,458,535).
  • an INVADER® operating system provides a method for detecting and quantifying DNA and RNA.
  • the INVADER® OS is based on a "perfect match" enzyme-substrate reaction.
  • the INVADER® OS uses proprietary CLEAVASE® enzymes (Third Wave Technologies, Inc (Madison, WI)), which recognize and cut only the specific structure formed during the INVADER® process which structure differs between the different alleles selected for detection, i.e. the disease-causing allele and the normal allele as well as between the different selected SNPs. Unlike the PCR- based methods, the INVADER® OS relies on linear amplification of the signal generated by the INVADER® process, rather than on exponential amplification of the target.
  • the detection of the enhanced or amplified nucleic acids representing the different alleles is performed using matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometric (MS) analysis described in the Examples below.
  • MALDI-TOF matrix-assisted laser desorption ionization/time-of-flight
  • MS mass spectrometric
  • a haplotyping method can be used for the purpose of the invention.
  • a halotyping method is a physical separation of alleles by cloning, followed by sequencing.
  • Other methods of haplotyping include, but are not limited to monoallelic mutation analysis (MAMA) (Papadopoulos et al. (1995) Nature Genet. 11 :99-102) and carbon nanotube probes (Woolley et al. (2000) Nature Biotech. 18:760-763).
  • MAMA monoallelic mutation analysis
  • U.S. Patent Application No. US 2002/0081598 also discloses a useful haplotying method which involves the use of PCR amplification.
  • Examples of newly discovered methods include for example, but are not limited to; SNP mapping (Davis et al, Methods Mol Biology, 2006; 351;75-92); Nanogen Nano Chip, (keen-Kim et al, 2006; Expert Rev Mol Diagnostic, 6;287-294); Rolling circle amplification (RCA) combined with circularable oligonucleotide probes (c-probes) for the detection of nucleic acids (Zhang et al, 2006: 363;61-70), luminex XMAP system for detecting multiple SNPs in a single reaction vessel (Dunbar SA, Clin Chim Acta, 2006;
  • Methods used to detect point mutations include denaturing gradient gel
  • DGGE restriction fragment length polymorphism analysis
  • RFLP restriction fragment length polymorphism analysis
  • chemical or enzymatic cleavage methods direct sequencing of target regions amplified by PCR (see above), single strand conformation polymorphism analysis (“SSCP”) and other methods well known in the art.
  • SSCP single strand conformation polymorphism analysis
  • One method of screening for point mutations is based on RNase cleavage of base pair mismatches in RNA/DNA or RNA/RNA heteroduplexes.
  • mismatch is defined as a region of one or more unpaired or mispaired nucleotides in a double-stranded RNA/RNA, RNA/DNA or DNA/DNA molecule. This definition thus includes mismatches due to insertion/deletion mutations, as well as single or multiple base point mutations.
  • protection from cleavage agents can be used to detect mismatched bases in RNA/RNA DNA/DNA, or RNA/DNA heteroduplexes (see, e.g., Myers et al. (1985) Science 230: 1242).
  • cleavage agents such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine
  • cleavage agents such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine
  • mismatched bases in RNA/RNA DNA/DNA or RNA/DNA heteroduplexes (see, e.g., Myers et al. (1985) Science 230: 1242).
  • the technique of "mismatch cleavage” starts by providing heteroduplexes formed by hybridizing a control nucleic acid, which is optionally labeled, e.g., RNA or DNA, comprising a nucleotide sequence of the allelic variant of the gene
  • RNA or DNA obtained from a tissue sample.
  • the double- stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as duplexes formed based on basepair mismatches between the control and sample strands.
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digest the mismatched regions.
  • either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions.
  • control and sample nucleic acids After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine whether the control and sample nucleic acids have an identical nucleotide sequence or in which nucleotides they are different. See, for example, U. S. Patent No. 6,455,249, Cotton et al. (1988) Proc. Natl. Acad. Sci. USA
  • control or sample nucleic acid is labeled for detection.
  • U.S. Patent 4,946,773 describes an R aseA mismatch cleavage assay that involves annealing single-stranded DNA or R A test samples to an RNA probe, and subsequent treatment of the nucleic acid duplexes with RNaseA. For the detection of mismatches, the single-stranded products of the RNaseA treatment, electrophoretically separated according to size, are compared to similarly treated control duplexes. Samples containing smaller fragments (cleavage products) not seen in the control duplex are scored as positive.
  • alterations in electrophoretic mobility can be used to identify the particular allelic variant.
  • SSCP single strand conformation polymorphism
  • Single- stranded DNA fragments of sample and control nucleic acids are denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in eiectrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments can be labeled or detected with labeled probes.
  • the sensitivity of the assay can be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • RNA rather than DNA
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al.
  • Gel Migration Single strand conformational polymorphism (SSCP; M. Orita et al., Genomics 5:8 74-8 79 (1989); Humphries et al, In: Molecular Diagnosis of Genetic Diseases, R. Elles, ed. pp321-340 (1996)) and temperature gradient gel electrophoresis (TGGE; R.M. Wartell et al, Nucl. Acids Res. 18:2699-2706 (1990)) are examples of suitable gel migration- based methods for determining the identity of a polymorphic site.
  • SSCP a single strand of DNA will adopt a conformation that is uniquely dependent of its sequence composition. This conformation is usually different, if even a single base is changed.
  • SSCP can be utilized to identify polymorphic sites, as wherein amplified products (or restriction fragments thereof of the target polynucleotide are denatured, then run on a non- denaturing gel. Alterations in the mobility of the resultant products are thus indicative of a base change. Suitable controls and knowledge of the "normal" migration patterns of the wild-type alleles can be used to identify polymorphic variants.
  • the identity of the allelic variant is obtained by analyzing the movement of a nucleic acid comprising the polymorphic region in
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for, example by adding a GC clamp of approximately 40 bp of high-melting GC rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing agent gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265: 1275).
  • multiplex PCR procedures using allele-specific primers can be used to simultaneously amplify multiple regions of a target nucleic acid (PCT).
  • PCT target nucleic acid
  • nucleic acid amplification procedures include transcription- based amplification systems (Malek, L.T. et al, U.S. Patent 5,130,238; Davey, C. et al, European Patent Application 329,822; Schuster et al.) U.S. Patent 5,169, 766; Miller, H.I.
  • Probes can be affixed to surfaces for use as “gene chips.” Such gene chips can be used to detect genetic variations by a number of techniques known to one of skill in the art. In one technique, oligonucleotides are arrayed on a gene chip for determining the DNA sequence of a by the sequencing by hybridization approach, such as that outlined in U.S. Patent Nos. 6,025,136 and 6,018,041. The probes of the present invention also can be used for fluorescent detection of a genetic sequence. Such techniques have been described, for example, in U.S. Patent Nos. 5,968,740 and 5,858,659.
  • a probe also can be affixed to an electrode surface for the electrochemical detection of nucleic acid sequences such as described by Kayyem et al. U.S. Patent No. 5,952,172 and by Kelley, S.O. et al. (1999) Nucleic Acids Res. 27:4830-4837.
  • the probe affixed to the surface of "gene chip” comprises a nucleotide sequence substantially complementary to: (i) GGTCCTCGCCTCC (SEQ ID NO: 2); (ii) GGTCCTAGCCTCC (SEQ ID NO: 3); (iii) GTTTGCCGTGTCG (SEQ ID NO: 4); (iv) GTTTGCTGTGTCG (SEQ ID NO: 5); (v) TCCCAAACTCCTG (SEQ ID NO: 6); (vi) TCCCAACCTCCTG (SEQ ID NO: 7); or (vii) any combinations of (i)-(vi).
  • the probe affixed to the surface of "gene chip” comprises a nucleotide sequence selected from (i) GGTCCTCGCCTCC (SEQ ID NO: 2); (ii)
  • GGTCCTAGCCTCC (SEQ ID NO: 3); (iii) GTTTGCCGTGTCG (SEQ ID NO: 4); (iv) GTTTGCTGTGTCG (SEQ ID NO: 5); (v) TCCCAAACTCCTG (SEQ ID NO: 6); (vi) TCCCAACCTCCTG (SEQ ID NO: 7); or (vii) any combinations of (i)-(vi).
  • biological sample denotes a sample taken or isolated from a biological organism, e.g., tissue cell culture supernatant, cell lysate, a homogenate of a tissue sample from a subject or a fluid sample from a subject.
  • Exemplary biological samples include, but are not limited to, blood, sputum, urine, spinal fluid, pleural fluid, nipple aspirates, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, feces, sperm, cells or cell cultures, serum, leukocyte fractions, smears, tissue samples of all kinds, embryos, etc.
  • the term also includes both a mixture of the above- mentioned samples such as whole human blood containing mycobacteria as well as food samples that contain free or bound nucleic acids or cells containing nucleic acids.
  • biological sample also includes untreated or pretreated (or pre-processed) biological samples.
  • a "biological sample” can contain cells from subject, but the term can also refer to non-cellular biological material, such as non-cellular fractions of blood, saliva, or urine, that can be used to measure gene expression levels or determine SNPs.
  • the sample is from a resection, biopsy, or core needle biopsy.
  • fine needle aspirate samples can be used. Samples can be either paraffin-embedded or frozen tissue.
  • the sample can be obtained by removing a sample of cells from a subject, but can also be accomplished by using previously isolated cells (e.g. isolated by another person).
  • the biological sample can be freshly collected or a previously collected sample.
  • the biological sample can be utilized for the detection of the presence and/or quantitative level of a biomolecule of interest.
  • Representative biomolecules include, but are not limited to, DNA, RNA, mRNA, polypeptides, and derivatives and fragments thereof.
  • the biological sample can be used for SNP determination for diagnosis of a diease or a disorder, e.g., Alzheimer's disease, using the methods, assays and systems of the invention.
  • biological sample can be a biological fluid.
  • biological fluids include, but are not limited to, saliva, bone marrow, blood, serum, plasma, urine, sputum, cerebrospinal fluid, an aspirate, tears, and any combinations thereof.
  • the biological sample is an untreated biological sample.
  • untreated biological sample refers to a biological sample that has not had any prior sample pre-treatment except for dilution and/or suspension in a solution.
  • Exemplary methods for treating a biological sample include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freezing and thawing, and any combinations thereof.
  • the biological sample is a frozen biological sample, e.g., a frozen tissue or fluid sample such as urine, blood, serum or plasma.
  • the frozen sample can be thawed before employing methods, assays and systems of the invention. After thawing, a frozen sample can be centrifuged before being subjected to methods, assays and systems of the invention.
  • the biological fluid sample can be treated with at least one chemical reagent, such as a protease inhibitor.
  • the biological fluid sample is a clarified biological fluid sample, for example, by centrifugation and collection of a supernatant comprising the clarified biological fluid sample.
  • a biological sample is a pre-processed biological sample, for example, supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, filtration, sonication, homogenization, lysis, thawing, amplification, purification, restriction enzyme digestion ligation and any combinations thereof.
  • a biological sample can be a nucleic acid product amplified after polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the biological sample can be treated with a chemical and/or biological reagent.
  • Chemical and/or biological reagents can be employed to protect and/or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing.
  • biomolecules e.g., nucleic acid and protein
  • One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of protein during processing.
  • chemical and/or biological reagents can be employed to release nucleic acid or protein from the sample.
  • the disclosure provides a computer system comprising: (a) a determination module configured to identify and detect at least one single nucleotide polymorphism (SNP) in a biological sample of a subject, wherein the SNP is selected from: (i) SNPl genotype A/A or AJC (or T/T or T/G in the complement) of SEQ ID NO: 1 , wherein SNPl is identified by rs277472 on SEQ ID NO: 1 , wherein SEQ ID NO.
  • SNP single nucleotide polymorphism
  • SNP2 is position 132,006,366 of SEQ ID NO: 1 identified by rsl0236235, wherein SEQ ID NO.
  • a storage module configured to store output data from the determination module;
  • a computing module adapted to identify from the output data at least one of AD risk associated alleles is present in the output data stored on the storage module; and
  • a display module for displaying if any of the AD risk associated alleles was identified or not, and/or displaying the detected alleles.
  • the computer system can include: (a) at least one memory containing at least one computer program adapted to control the operation of the computer system to implement a method that includes: (i) receiving data of the level of expression or intensity of signal of measured for a PLXNA4 isoform (e.g. TS1 or TS3) mRNA ; (ii) generating a report of intensity of expression or intensity of signal of measured PLXNA4 isoform mRNA in a biological sample and optionally a reference level for PLXNA4 isoform mRNA signal intensity; and (b) at least one processor for executing the computer program.
  • a PLXNA4 isoform e.g. TS1 or TS3
  • a computer system can include: (a) at least one memory containing at least one computer program adapted to control the operation of the computer system to implement a method that includes: (i) receiving data of the level of expression or intensity of signal of measured AD risk associated allele levels (ii) generating a report of intensity of expression or intensity of signal of measured AD risk associated allele levels in a biological sample and optionally a reference level AD risk associated allele signal intensity; and (b) at least one processor for executing the computer program.
  • a computer system can include, for example, an Intel or AMD x86 based single or multi-core central processing unit (CPU), an ARM processor or similar computer processor for processing the data.
  • the CPU or microprocessor can be any conventional general purpose single-or multi-chip microprocessor such as an Intel and AMD processor, a SPARC processor, or an ARM processor.
  • the microprocessor may be any conventional or special purpose microprocessor such as a digital signal processor or a graphics processor.
  • the microprocessor typically has conventional address lines,
  • the software according to the invention can be executed on dedicated system or on a general purpose computer having a DOS, CPM, Windows, Unix, Linix or other operating system.
  • the system can include non-volatile memory, such as disk memory and solid state memory for storing computer programs, software and data and volatile memory, such as high speed ram for executing programs and software.
  • Computer-readable physical storage media useful in various embodiments of the invention can include any physical computer-readable storage medium, e.g. , solid state memory (such as flash memory), magnetic and optical computer-readable storage media and devices, and memory that uses other persistent storage technologies.
  • a computer readable media can be any tangible media that allows computer programs and data to be accessed by a computer.
  • Computer readable media can include volatile and nonvolatile, removable and non-removable tangible media implemented in any method or technology capable of storing information such as computer readable instructions, program modules, programs, data, data structures, and database information.
  • computer readable media includes, but is not limited to, RAM (random access memory), ROM (read only memory), EPROM (erasable programmable read only memory), EEPROM (electrically erasable programmable read only memory), flash memory or other memory technology, CD-ROM (compact disc read only memory), DVDs (digital versatile disks), Blue-ray, USB drives, micro-SD drives, or other optical storage media, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage media, other types of volatile and non- volatile memory, and any other tangible medium which can be used to store information and which can read by a computer including and any suitable combination of the foregoing.
  • RAM random access memory
  • ROM read only memory
  • EPROM erasable programmable read only memory
  • EEPROM electrically erasable programmable read only memory
  • flash memory or other memory technology CD-ROM (compact disc read only memory), DVDs (digital versatile disks), Blue-ray, USB drives, micro-SD drives, or other optical storage media, magnetic cassettes, magnetic tape
  • the present invention can be implemented on a stand-alone computer or as part of a networked computer system.
  • all the software and data can reside on local memory devices, for example an optical disk or flash memory device can be used to store the computer software for implementing the invention as well as the data.
  • the software or the data or both can be accessed through a network connection to remote devices.
  • the invention can use a client-server environment over a network, e.g., a public network such as the internet or a private network to connect to data and resources stored in remote and/or centrally located locations.
  • a server such as a web server can provide access, e.g.
  • a client computer executing a client software or program, such as a web browser, connects to the server over the network.
  • the client software provides a user interface for a user of the invention to input data and information and receive access to data and information.
  • the client software can be viewed on a local computer display or other output device and can allow the user to input information, such as by using a computer keyboard, mouse or other input device.
  • the server executes one or more computer programs that receives data input through the client software, processes data according to the invention and outputs data to the user, as well as provide access to local and remote computer resources.
  • the user interface can include a graphical user interface comprising an access element, such as a text box, that permits entry of data from the assay, e.g. , the data from a positive reference cancer cell, as well as a display element that can provide a graphical read out of the results of a comparison with a cancer cell with a known metastatic potential or invasive capacity, or data sets transmitted to or made available by a processor following execution of the instructions encoded on a computer-readable medium.
  • an access element such as a text box
  • a display element that can provide a graphical read out of the results of a comparison with a cancer cell with a known metastatic potential or invasive capacity, or data sets transmitted to or made available by a processor following execution of the instructions encoded on a computer-readable medium.
  • Embodiments of the invention also provide for systems (and computer readable medium providing instructions for causing computer systems) to perform a method for determining quality assurance of a pluripotent stem cell population according to the methods as disclosed herein.
  • the computer system software can include one or more functional modules, which can be defined by computer executable instructions recorded on computer readable media and which cause a computer to perform, when executed, a method according to one or more embodiments of the invention.
  • the modules can be segregated by function for the sake of clarity, however, it should be understood that the modules need not correspond to discreet blocks of code and the described functions can be carried out by the execution of various software code portions stored on various media and executed at various times.
  • the modules can perform other functions, thus the modules are not limited to having any particular function or set of functions.
  • functional modules are, for example, but are not limited to, an array module, a determination module, a storage module, a reference
  • the functional modules can be executed using one or multiple computers, and by using one or multiple computer networks.
  • the information embodied on one or more computer-readable media can include data, computer software or programs, and program instructions, which as a result of being executed by a computer, transform the computer to special purpose machine and can cause the computer to perform one or more of the functions described herein.
  • Such instructions can be originally written in any of a plurality of programming languages, for example, Java, J#, Visual Basic, C, C#, C++, Fortran, Pascal, Eiffel, Basic, COBOL assembly language, and the like, or any of a variety of combinations thereof.
  • the computer-readable media on which such instructions are embodied can reside on one or more of the components of a computer system or a network of computer systems according to the invention.
  • a computer-readable media can be transportable such that the instructions stored thereon can be loaded onto any computer resource to implement the aspects of the present invention discussed herein.
  • the instructions stored on computer readable media are not limited to instructions embodied as part of an application program running on a host computer. Rather, the instructions may be embodied as any type of computer code (e.g., object code, software or microcode) that can be employed to program a computer to implement aspects of the present invention.
  • the computer executable instructions may be written in a suitable computer language or combination of several languages.
  • the system as disclosed herein can be configured to receive data from an automated protein analysis systems, for example, using immunoassay, for example western blot analysis or ELISA, or a high through-put protein detection method, for example but are not limited to automated
  • immunohistochemistry apparatus for example, robotically automated immunodetection apparatus which in an automated system can perform immunohistochemistry procedure and detect intensity of immunostaining, such as intensity of an antibody staining of the substrates and produce output data.
  • automated immunohistochemistry apparatus are commercially available, and can be readily adapted to automatically detect the level of protein expression in the assay as disclosed herein, and include, for example but not limited to such Autostainers 360, 480, 720 and Labvision PT module machines from Lab Vision Corporation, which are disclosed in U.S. Patents 7,435,383; 6,998,270; 6,746,851, 6,735,531; 6,349,264; and 5,839; 091 which are incorporated herein in their entirety by reference.
  • Other commercially available automated immunohistochemistry instruments are also encompassed for use in the present invention, for example, but not are limited BONDTM Automated
  • Immunohistochemistry & In Situ Hybridization System Automate slide loader from GTI vision. Automated analysis of immunohistochemistry can be performed by commercially available systems such as, for example, IHC Scorer and Path EX, which can be combined with the Applied spectral Images (ASI) CytoLab view, also available from GTI vision or Applied Spectral Imaging (ASI) which can all be integrated into data sharing systems such as, for example, Laboratory Information System (LIS), which incorporates Picture Archive Communication System (PACS), also available from Applied Spectral Imaging (ASI) (see world- wide -web: spectral-imaging.com).
  • LIS Laboratory Information System
  • PES Picture Archive Communication System
  • ASI Applied Spectral Imaging
  • Other a determination module can be an automated immunohistochemistry systems such as NexES® automated immunohistochemistry (IHC) slide staining system or BenchMark® LT automated IHC instrument from Ventana
  • BioGenex Super Sensitive MultiLink® Detection Systems in either manual or automated protocols can also be used as the detection module, preferably using the BioGenex Automated Staining Systems.
  • Such systems can be combined with a BioGenex automated staining systems, the ⁇ 6000TM (and its predecessor, the OptiMax® Plus), which is geared for the Clinical Diagnostics lab, and the GenoMx 6000TM, for Drug Discovery labs.
  • Both systems BioGenex systems perform "All-in-One, All-at-Once" functions for cell and tissue testing, such as Immunohistochemistry (IHC) and In Situ Hybridization (ISH).
  • IHC Immunohistochemistry
  • ISH In Situ Hybridization
  • a system as disclosed herein can receive data of intensity of protein expression of PLXNA4 from an automated ELISA system (e.g. DSX® or DS2® form Dynax, Chantilly, VA or the ENEASYSTEM III®, Triturus®, The Mago® Plus);
  • an automated ELISA system e.g. DSX® or DS2® form Dynax, Chantilly, VA or the ENEASYSTEM III®, Triturus®, The Mago® Plus
  • Densitometers e.g. X-Rite-508-Spectro Densitometer®, The HYRYSTM 2 densitometer
  • automated Fluorescence in situ hybridization systems see for example, United States Patent 6, 136,540
  • 2D gel imaging systems coupled with 2-D imaging software microplate readers
  • Fluorescence activated cell sorters FACS
  • FACS Fluorescence activated cell sorters
  • radio isotope analyzers e.g. scintillation counters
  • a system as disclosed herein can receive data can receive data of intensity of mRNA expression of PLXNA4 (e.g., isoform TS l or TS 3) or presence of absence of an AD risk associated allele from any method of determining gene or nucleic acid expression or mutations or SNP genotyping.
  • the system as disclosed herein can be configured to receive data from an automated gene expression analysis system, e.g., an automated protein expression analysis including but not limited Mass Spectrometry systems including MALDI-TOF, or Matrix Assisted Laser Desorption Ionization - Time of Flight systems; SELDI-TOF-MS ProteinChip array profiling systems, e.g.
  • an automated gene expression analysis system can record the data electronically or digitally, annotated and retrieved from databases including, but not limited to GenBank (NCBI) protein and DNA databases such as genome, ESTs, SNPS, Traces, Celara, Ventor Reads, Watson reads, HGTS, etc.; Swiss Institute of Bioinformatics databases, such as ENZYME, PROSITE, SWISS-2DPAGE, Swiss-Prot and TrEMBL databases; the Melanie software package or the ExPASy WWW server, etc., the SWISS-MODEL, Swiss-Shop and other network-based computational tools; the Comprehensive Microbial Resource database (The institute of Genomic Research).
  • the resulting information can be stored in a relational data base that may be employed to determine homologies between the reference data or genes or proteins within and among genomes.
  • a system as disclosed herein can receive data from an allele-specific PCR.
  • allele-specific PCR refers to PCR techniques where the primer pairs are chosen such that amplification is dependent upon the input template nucleic acid containing the polymorphism of interest.
  • primer pairs are chosen such that at least one primer is an allele- specific oligonucleotide primer.
  • allele-specific primers are chosen so that
  • amplification creates a restriction site, facilitating identification of a polymorphic site.
  • amplification of the target polynucleotide is by multiplex PCR (Wallace et al. (PCT Application W089/10414)). Through the use of multiplex PCR, a multiplicity of regions of a target polynucleotide can be amplified simultaneously. This is particularly advantageous in embodiments where more than one SNP is to be detected.
  • multiplex PCR procedures using allele-specific primers can be used to simultaneously amplify multiple regions of a target nucleic acid (PCT).
  • PCT target nucleic acid
  • nucleic acid amplification procedures include transcription- based amplification systems (Malek, L.T. et al, U.S. Patent 5,130,238; Davey, C. et al, European Patent Application 329,822; Schuster et al.) U.S. Patent 5,169, 766; Miller, H.I. et al, PCT - Application W089/06700; Kwoh, D. et al, Proc. Natl. Acad Sci. (U.S.A) 86: 1173 Z1989); Gingeras, T.R. et al., PCT Application W088/10315)), or isothermal amplification methods (Walker, G.T. et al, Proc. Natl. 4cad Sci. (U.S.A) 89:392-396 (1992)) can also be used.
  • a system as disclosed herein can receive data from any genotyping assay known by persons of ordinary skill in the art, including, but not limted to, those disclosed in U.S. Patent No. 6,472,157; U.S. Patent Application Publications
  • the data can be received from a memory, a storage device, or a database.
  • the memory, storage device or database can be directly connected to the computer system retrieving the data, or connected to the computer through a wired or wireless connection technology and retrieved from a remote device or system over the wired or wireless connection. Further, the memory, storage device or database, can be located remotely from the computer system from which it is retrieved.
  • connection technologies for use with the present invention include, for example parallel interfaces (e.g., PAT A), serial interfaces (e.g., SATA, USB, Firewire,), local area networks (LAN), wide area networks (WAN), Internet, Intranet, and Extranet, and wireless (e.g., Blue Tooth, Zigbee, WiFi, WiMAX, 3G, 4G) communication technologies
  • Storage devices are also commonly referred to in the art as "computer-readable physical storage media" which is useful in various embodiments, and can include any physical computer-readable storage medium, e.g., magnetic and optical computer-readable storage media, among others. Carrier waves and other signal-based storage or transmission media are not included within the scope of storage devices or physical computer-readable storage media encompassed by the term and useful according to the invention.
  • the storage device is adapted or configured for having recorded thereon cytokine level information. Such information can be provided in digital form that can be transmitted and read electronically, e.g., via the Internet, on diskette, via USB (universal serial bus) or via any other suitable mode of communication.
  • stored refers to a process for recording information, e.g., data, programs and instructions, on the storage device that can be read back at a later time.
  • information e.g., data, programs and instructions
  • Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to contribute to the data of (i) the level of expression of a PLXNA4 isoform (TS1 or TS3) mRNA and/or (ii) presence or absence of an AD risk associated allele as disclosed in the methods herein.
  • a variety of software programs and formats can be used to store information on the storage device. Any number of data processor structuring formats (e.g., text file or database) can be employed to obtain or create a medium having recorded scorecard thereon.
  • data processor structuring formats e.g., text file or database
  • the system has a processor for running one or more programs, e.g., where the programs can include an operating system (e.g., UNIX, Windows), a relational database management system, an application program, and a World Wide Web server program.
  • the application program can be a World Wide Web application that includes the executable code necessary for generation of database language statements (e.g., Structured Query Language (SQL) statements).
  • SQL Structured Query Language
  • the executable can include embedded SQL statements.
  • the World Wide Web application can include a configuration file which contains pointers and addresses to the various software entities that provide the World Wide Web server functions as well as the various external and internal databases which can be accessed to service user requests.
  • the Configuration file can also direct requests for server resources to the appropriate hardware devices, as may be necessary should the server be distributed over two or more separate computers.
  • the World Wide Web server supports a TCP/IP protocol.
  • Local networks such as this are sometimes referred to as "Intranets."
  • An advantage of such Intranets is that they allow easy communication with public domain databases residing on the World Wide Web (e.g., the GenBank or Swiss Pro World Wide Web site).
  • users can directly access data (via Hypertext links for example) residing on Internet databases using a HTML interface provided by Web browsers and Web servers.
  • the system as disclosed herein can be used to compare the data of intensity of one or more of (i) the level of expression of PLXNA4 mR A (e.g.
  • reference data e.g., reference intensity values
  • the system can compare the data in a "comparison module" which can use a variety of available software programs and formats for the comparison operative to compare sequence information determined in the determination module to reference data.
  • the comparison module is configured to use pattern recognition techniques to compare levels of expression (e.g., mRNA levels and/or protein levels) as well as compare sequence information (e.g., identify the presence of different SNPs of AD risk associated alleles from one or more entries to one or more reference data patterns.
  • the comparison module can be configured using existing commercially-available or freely-available software for comparing patterns, and may be optimized for particular data comparisons that are conducted.
  • the comparison module can also provide computer readable information related to the level or amount of intensity of expression of the level of expression of a specific PLXNA4 isoform (e.g., TS l or TS3) mRNA.
  • the comparison module provides computer readable comparison result that can be processed in computer readable form by predefined criteria, or criteria defined by a user, to provide a report which comprises content based in part on the comparison result that may be stored and output as requested by a user using a display module.
  • a display module enables display of a content based in part on the comparison result for the user, wherein the content is a report indicative of the results of the comparison of the intensity of expression of (i) the level of expression of a PLXNA4 isoform mRNA; and/or (ii) presence of an AD risk associated allele with their respective reference values.
  • the display module enables display of a report or content based in part on the comparison result for the end user, wherein the content is a report indicative of the results of the comparison of the intensity of expression of any one or more of (i) the level of expression of PLXNA4 isoform (e.g., TS 1 or TS2) mRNA; and/or (ii) presence of an AD risk associated allele.
  • the content is a report indicative of the results of the comparison of the intensity of expression of any one or more of (i) the level of expression of PLXNA4 isoform (e.g., TS 1 or TS2) mRNA; and/or (ii) presence of an AD risk associated allele.
  • the comparison module can include an operating system (e.g., UNIX, Windows) on which runs a relational database management system, a World Wide Web application, and a World Wide Web server.
  • World Wide Web application can includes the executable code necessary for generation of database language statements [e.g., Standard Query Language (SQL) statements].
  • the executables can include embedded SQL statements.
  • the World Wide Web application may include a configuration file which contains pointers and addresses to the various software entities that comprise the server as well as the various external and internal databases which must be accessed to service user requests.
  • the Configuration file also directs requests for server resources to the appropriate hardware—as may be necessary should the server be distributed over two or more separate computers.
  • the World Wide Web server supports a TCP/IP protocol. Local networks such as this are sometimes referred to as "Intranets.” An advantage of such Intranets is that they allow easy
  • World Wide Web e.g., the GenBank or Swiss Pro World Wide Web site.
  • users can directly access data (via Hypertext links for example) residing on Internet databases using an HTML interface provided by Web browsers and Web servers.
  • HTML interface provided by Web browsers and Web servers.
  • other interfaces such as HTTP, FTP, SSH and VPN based interfaces can be used to connect to the Internet databases.
  • a computer-readable media can be transportable such that the instructions stored thereon, such as computer programs and software, can be loaded onto any computer resource to implement the aspects of the present invention discussed herein.
  • the instructions stored on the computer-readable medium, described above are not limited to instructions embodied as part of an application program running on a host computer. Rather, the instructions may be embodied as any type of computer code (e.g., software or microcode) that can be employed to program a processor to implement aspects of the present invention.
  • the computer executable instructions can be written in a suitable computer language or combination of several languages. Basic computational biology methods are described in, e.g. Setubal and Meidanis et al,
  • the computer instructions can be implemented in software, firmware or hardware and include any type of programmed step undertaken by modules of the information processing system.
  • the computer system can be connected to a local area network (LAN) or a wide area network (WAN).
  • LAN local area network
  • WAN wide area network
  • the local area network can be a corporate computing network, including access to the Internet, to which computers and computing devices comprising the data processing system are connected.
  • the LAN uses the industry standard Transmission Control Protocol/Internet Protocol (TCP/IP) network protocols for communication.
  • TCP Transmission Control Protocol Transmission Control Protocol
  • TCP can be used as a transport layer protocol to provide a reliable, connection-oriented, transport layer link among computer systems.
  • the network layer provides services to the transport layer.
  • TCP provides the mechanism for establishing, maintaining, and terminating logical connections among computer systems.
  • TCP transport layer uses IP as its network layer protocol.
  • TCP provides protocol ports to distinguish multiple programs executing on a single device by including the destination and source port number with each message.
  • TCP performs functions such as transmission of byte streams, data flow definitions, data acknowledgments, lost or corrupt data re-transmissions, and multiplexing multiple connections through a single network connection.
  • TCP is responsible for encapsulating information into a datagram structure.
  • the LAN can conform to other network standards, including, but not limited to, the International Standards Organization's Open Systems Interconnection, IBM's SNA, Novell's Netware, and Banyan VINES.
  • the computer system as described herein can include any type of electronically connected group of computers including, for instance, the following networks: Internet, Intranet, Local Area Networks (LAN) or Wide Area Networks (WAN).
  • the connectivity to the network may be, for example, remote modem, Ethernet (IEEE 802.3), Token Ring (IEEE 802.5), Fiber Distributed Datalink Interface (FDDI) or Asynchronous Transfer Mode (ATM).
  • the computing devices can be desktop devices, servers, portable computers, hand-held computing devices, smart phones, set-top devices, or any other desired type or configuration.
  • a network includes one or more of the following, including a public internet, a private internet, a secure internet, a private network, a public network, a value-added network, an intranet, an extranet and combinations of the foregoing.
  • a comparison module provides computer readable data that can be processed in computer readable form by predefined criteria, or criteria defined by a user, to provide a retrieved content that may be stored and output as requested by a user using a display module.
  • the computerized system can include or be operatively connected to an output module.
  • the output module is a display module, such as computer monitor, touch screen or video display system.
  • the display module allows user instructions to be presented to the user of the system, to view inputs to the system and for the system to display the results to the user as part of a user interface.
  • the computerized system can include or be operative connected to a printing device for producing printed copies of information output by the system.
  • the results can be displayed on a display module or printed in a report, e.g. , a to indicate any one or more of (i) the level of expression of a PLXNA4 isoform (e.g., TS1 or TS3) mRNA; and/or (ii) presence of at least one AD risk associated allele.
  • the report is a hard copy printed from a printer.
  • the computerized system can use light or sound to report the result.
  • the report produced by the methods, assays, systems and kits as disclosed herein can comprise a report which is color coded to signal or indicate any one or more of (i) the level of expression of a PLXNA4 (eg., isoform TS1 or TS3) mRNA; and/or (ii) presence of at least one AD risk associated allele.
  • a PLXNA4 eg., isoform TS1 or TS3
  • the report can also present text, either verbally or written, giving a recommendation of if a subject is amenable to treatment with a TS1 PLXNA4 inhibitory agent as disclosed herein.
  • the report provides just values or numerical scores for the presence of any one or more of the (i) the level of expression of PLXNA4 mRNA; and/or (ii) presence of at least one AD risk associated allele which can be readily compared by a physician with reference values as disclosed herein.
  • the report data from the comparison module can be displayed on a computer monitor as one or more pages of the printed report.
  • a page of the retrieved content can be displayed through printable media.
  • the display module can be any device or system adapted for display of computer readable information to a user.
  • the display module can include speakers, cathode ray tubes (CRTs), plasma displays, light-emitting diode (LED) displays, liquid crystal displays (LCDs), printers, vacuum florescent displays (VFDs), surface-conduction electron- emitter displays (SEDs), field emission displays (FEDs), etc.
  • a World Wide Web browser can be used to provide a user interface to allow the user to interact with the system to input information, construct requests and to display retrieved content.
  • the various functional modules of the system can be adapted to use a web browser to provide a user interface.
  • a Web browser Using a Web browser, a user can construct requests for retrieving data from data sources, such as data bases and interact with the comparison module to perform comparisons and pattern matching.
  • the user can point to and click on user interface elements such as buttons, pull down menus, scroll bars, etc. conventionally employed in graphical user interfaces to interact with the system and cause the system to perform the methods of the invention.
  • the requests formulated with the user's Web browser can be transmitted over a network to a Web application that can process or format the request to produce a query of one or more database that can be employed to provide the pertinent information.
  • PLXNA4 is a viable target for therapeutic treatment of AD. Accordingly, provided herein is a method for treatment of AD in a subject. Generaly the method comprises administering to the subject a pharmaceutically acceptable composition comprising a TS 1 PLXNA4 inhibitory agent.
  • Subjects amenable to methods of treatment are subjects that have been diagnosed with Alzheimer's disease. Methods for diagnosing Alzheimer's disease are well known in the art.
  • stage of Alzheimer's disease can be assessed using the Functional Assessment Staging (FAST) scale, which divides the progression of Alzheimer's disease into 16 successive stages under 7 major headings of functional abilities and losses:
  • Stage 1 is defined as a normal adult with no decline in function or memory.
  • Stage 2 is defined as a normal older adult who has some personal awareness of functional decline, typically complaining of memory deficit and forgetting the names of familiar people and places.
  • Stage 3 (early Alzheimer's disease) manifests symptoms in demanding job situation, and is characterized by disorientation when traveling to an unfamiliar location; reports by colleagues of decreased performance; name- and word-finding deficits; reduced ability to recall information from a passage in a book or to remember a name of a person newly introduced to them; misplacing of valuable objects; decreased concentration.
  • FAST Functional Assessment Staging
  • stage 4 the patient may require assistance in complicated tasks such as planning a party or handling finances, exhibits problems remembering life events, and has difficulty concentrating and traveling.
  • stage 5 the patient requires assistance to perform everyday tasks such as choosing proper attire. Disorientation in time, and inability to recall important information of their current lives, occur, but patient can still remember major information about themselves, their family and others.
  • stage 6 the patient begins to forget significant amounts of information about themselves and their surroundings and require assistance dressing, bathing, and toileting. Urinary incontinence and disturbed patterns of sleep occur. Personality and emotional changes become quite apparent, and cognitive abulia is observed.
  • stage 7 severe Alzheimer's disease
  • speech ability becomes limited to just a few words and intelligible vocabulary may be limited to a single word. A patient can lose the ability to walk, sit up, or smile, and eventually cannot hold up the head.
  • AD Alzheimer's disease
  • cellular and molecular testing methods disclosed in US Patent No.: US 7771937, US 7595167, US 55580748, and PCT Application No.: WO2009/009457, the content of which is incorporated by reference in its entirety.
  • protein-based biomarkers for AD some of which can be detected by non-invasive imaging, e.g., PET, are disclosed in US 7794948, the content of which is incorporated by reference in its entirety.
  • AD risk genes increase the risk of developing AD.
  • ⁇ - ⁇ 4 apolipoprotein ⁇ - ⁇ 4
  • ⁇ - ⁇ 4 is one of three common forms, or alleles, of the APOE gene; the others are APOE-e2 and APOE-e3.
  • APOE provides the blueprint for one of the proteins that carries cholesterol in the bloodstream.
  • AD risk genes in addition to APOE-e4 are well established in the art. Some of them are disclosed in US Pat. App. No.: US 2010/0249107, US 2008/ 0318220, US 2003/0170678 and PCT Application No.: WO 2010/048497, the content of which is incorporated by reference in its entirety. Genetic tests are well established in the art and are available, for example for APOE- e4. A subject carrying the ⁇ - ⁇ 4 allele can, therefore, be identified as a subject at risk of developing AD.
  • subjects with ⁇ burden are amenable to the methods described herein.
  • Such subjects include, but not limited to, the ones with Down syndrome, Huntington disease, the unaffected carriers of APP or presenilin gene mutations, and the late onset AD risk factor, apolipoprotein ⁇ - ⁇ 4.
  • AD patients that are currently receiving other AD therapeutic treatment can also be subjected to the methods of treatment as described herein.
  • a subject who has been diagnosed with an increased risk for developing AD e.g., using the diagnostic methods and assays described herein or any AD diagnostic methods known in the art, can be subjected to the methods of treatment as described herein.
  • the subject with at least one AD risk-associated allele but no AD symptoms, including undetectable level of amyloid-beta protein in the brain and/or no detectable cognitive impairement can be administered with a preventive treatment.
  • prevention interventions include, but are not limited to life style advice, including e.g., prescribing an aerobic exercise regime to exercise the body and/or mental exercises to keep brain active, dietary advice, including increase in intake of omega-3 fatty acids, fruits and vegetables, fish or poultry, whole-grain breads and cereals, or reduction of sugar or cholesterol rich food intake to lower cholesterol, and administering pharmaceutical agents effective in prevention or treatment of AD.
  • the subject having at least one AD risk-associated allele and exhibiting AD symptons can be treated with the methods of treatment described herein.
  • the subject diagnosed with AD can be treated with a drug known in the art such as cholinesterase inhibitors (for example,
  • the term "treatment or prevention for AD” will encompass treating a subject diagnosed with AD to slow down or ameliorate at least one symptom associated with AD, or treating a subject with an increased risk for AD, e.g, carrying an AD risk associated allele described herein to avoid or delay the onset of AD.
  • prevention refers to a complete avoidance of symptoms, such as cognitive impairment or measurable markers of AD, level of ⁇ in the brain, or delay the onset of AD. Inhibition of AD development is also considered a preventive measure even if it does not confer a complete avoidance of AD symptons.
  • inhibiting refers to a reduced severity or degree of any one or more of those symptoms or markers, relative to those symptoms or markers arising in a control or non-treated individual with a similar likelihood or susceptibility of developing AD, or relative to symptoms or markers likely to arise based on historical or statistical measures of populations affected by AD.
  • reduced severity is meant at least about 20% in the severity or degree of a symptom or measurable marker, e.g., level of ⁇ in the brain, relative to a control, such as without administration of the treatment described herein, e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or even 100% (i.e., no or non-detectable level of cognitive impairment or measurable markers, e.g., ⁇ level).
  • kits for the practice of the assays and methods disclosed herein.
  • the kits preferably include one or more containers containing a TS1 PLXNA4 inhibitory agent and a pharmaceutically acceptable excipient.
  • the kit can optionally contain additional therapeutics to be co-administered with the TS1 PLXNA4 inhibitory agent.
  • the kit can comprise instructions for administration of the TS 1 PLXNA4 inhibitory agent to a subject with AD or at risk of AD.
  • the kits can also optionally include appropriate systems (e.g. opaque containers) or stabilizers (e.g. antioxidants) to prevent degradation of the TS1 PLXNA4 inhibitory agent by light or other adverse conditions.
  • the kit can comprise probes, e.g., allele-specific
  • the kit can comprise probes, e.g., allele-specific oligonucleotide probes or allele specific primer probes for detecting one or more of: (i) SNP1 genotype A/A or A/C (or T/T or T/G in the complement) of SEQ ID NO: 1, wherein SNP1 is identified by rs277472 on SEQ ID NO: 1, wherein SEQ ID NO.
  • SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of plexin A4 (PLXNA4); (ii) SNP2 genotype T/T or T/C (or A/A or A/C in the complement) of SEQ ID NO: 1, wherein SNP2 is position 132,006,366 of SEQ ID NO: 1 identified by rsl0236235, wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; (iii) SNP3 genotype C/C or C/A (or G/G or G/T in the complement) of SEQ ID NO: 1, wherein SNP3 identified by rsl 1761937 on SEQ ID NO: 1, wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; and (iv) any combinations thereof, for the practice of the methods of this invention.
  • PLXNA4 genomic nucleic acid sequence of plexin A4
  • Allele-specific probes are well know by persons of ordinary skill in the art, with oligonucleotides encompassed for use as probes, and refer to such as genomic DNA, mRNA, or other suitable sources of nucleic acid oligonucleotides. For such purposes, the
  • oligonucleotides must be capable of specifically hybridizing to a target polynucleotide or DNA nucleic acid molecule.
  • two nucleic acid molecules are said to be capable of specifically hybridizing to one another if the two molecules are capable of forming an anti-parallel, double-stranded nucleic acid structure under hybridizing conditions.
  • allele-specific oligonucleotide refers to an oligonucleotide that is able to hybridize to a region of a target polynucleotide spanning the sequence, mutation, or polymorphism being detected and is substantially unable to hybridize to a corresponding region of a target polynucleotide that either does not contain the sequence, mutation, or polymorphism being detected or contains an altered sequence, mutation, or polymorphism.
  • allele-specific is not meant to denote an absolute condition. Allele-specificity will depend upon a variety of environmental conditions, including salt and formamide concentrations, hybridization and washing conditions and stringency.
  • one or more allele- specific oligonucleotides can be employed for each target polynucleotide.
  • allele- specific oligonucleotides will be completely complementary to the target polynucleotide.
  • departures from complete complementarity are permissible.
  • an oligonucleotide In order for an oligonucleotide to serve as a primer oligonucleotide, however, it typically need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular environmental conditions employed. Establishing environmental conditions typically involves selection of solvent and salt concentration, incubation temperatures, and incubation times.
  • the ASO comprises a nucleotide sequence substantially complementary to: (i) GGTCCTCGCCTCC (SEQ ID NO: 2); (ii) GGTCCTAGCCTCC (SEQ ID NO: 3); (iii) GTTTGCCGTGTCG (SEQ ID NO: 4); (iv) GTTTGCTGTGTCG (SEQ ID NO: 5); (v) TCCCAAACTCCTG (SEQ ID NO: 6); (vi) TCCCAACCTCCTG (SEQ ID NO: 7); or (vii) any combinations of (i)-(vi).
  • the ASO comprises a nucleotide sequence selected from (i) GGTCCTCGCCTCC (SEQ ID NO: 2); (ii) GGTCCTAGCCTCC (SEQ ID NO: 3); (iii) GTTTGCCGTGTCG (SEQ ID NO: 4); (iv) GTTTGCTGTGTCG (SEQ ID NO: 5); (v) TCCCAAACTCCTG (SEQ ID NO: 6); (vi) TCCCAACCTCCTG (SEQ ID NO: 7); or (vii) any combinations of (i)-(vi).
  • the kit can be used to perform a genotyping assay used to determine the AD risk associated SNPs disclosed herien, where the genotyping assay is selected from any or a combination in the group consisting of: PCR-based assays, RT-PCR, nucleic acid hybridization, sequence analysis, TaqMan SNP genotyping probes, microarrays, direct or indirect sequencing, restriction site analysis, hybridization based genotyping assays, gel migration assays, antibodies assays, fluorescent polarization, mass spectroscopy, allele- specific PCR, single-strand conformational polymorphism (SSCP) analysis, heteroduplex analysis, oligonucleotide ligation, PCR-RFLP, allele-specific amplification (ASA), single- molecule dilution (SMD),coupled amplification and sequencing (CAS), Restriction enzyme analysis, restriction fragment length polymorphism (RFLP), ligation based assays, single base extension (or minisequencing), MALD
  • kits can optionally include instructional materials containing directions (i.e., protocols) providing for the use of a compounds and composition in the treatment of AD. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
  • Kits for determining if a subject is at increased risk of developing Alzheimer's disease will include at least one reagent specific for detecting for the presence or absence of the AD risk associated SNPs described herein or antibodies specific for detecting the gene expression products (e.g., PLXNA4) associated with AD risk associated SNPs, and instructions for observing that the subject is at increased risk of developing Alzheimer's disease if the presence of at least one of the SNPs described herein is detected.
  • the kit may optionally include a nucleic acid for detection of the gene of interest.
  • the diagnostic kit comprises (a) an antibody which binds PLXNA4 conjugated to a solid support and (b) a second antibody which binds PLXNA4 conjugated to a detectable group.
  • the reagents may also include ancillary agents such as buffering agents and protein stabilizing agents, e.g., polysaccharides and the like.
  • the diagnostic kit may further include, where necessary, other members of the signal-producing system of which system the detectable group is a member (e.g., enzyme substrates), agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like.
  • a second embodiment of a test kit comprises (a) an antibody as above, and (b) a specific binding partner for the antibody conjugated to a detectable group.
  • Ancillary agents as described above can likewise be included.
  • the test kit may be packaged in any suitable manner, typically with all elements in a single container along with a sheet of printed instructions for carrying out the test.
  • the diagnostic kits can comprise primers or probes for detection of mRNA level of PLXNA4 isoform TS1 and/or TS3.
  • a method for inhibiting progression of Alzheimer's disease comprising administering to a subject having or at risk for Alzheimer's disease a therapeutically effective amount of a TS 1 PLXNA4 inhibitory agent.
  • the method comprising administering to a subject determined to have one or more of AD risk associated single nucleotide polymorphism (SNP) selected from: (i) SNPl genotype A/A or A/C (or T/T or T/G in the complement) of SEQ ID NO: 1, wherein SNPl is identified by rs277472 on SEQ ID NO: 1, wherein SEQ ID NO.
  • SNP AD risk associated single nucleotide polymorphism
  • SNP2 genotype T/T or T/C (or A/A or A/C in the complement) of SEQ ID NO: 1, wherein SNP2 is position 132,006,366 of SEQ ID NO: 1 identified by rsl0236235, wherein SEQ ID NO.
  • a method for inhibiting or reducing neurofibrillary tangles in the brain comprising administering to a subject having or at risk for having neurofibrillary tangles in the brain a therapeutically effective amount of a TS1 PLXNA4 inhibitory agent.
  • a method for inhibiting or reducing neurofibrillary tangles in the brain of a subject in need thereof comprising administering to a subject determined to have one or more of AD risk associated single nucleotide polymorphism (SNP) selected from: (i) SNPl genotype A/A or A/C (or T/T or T/G in the complement) of SEQ ID NO: 1, wherein SNPl is identified by rs277472 on SEQ ID NO: 1, wherein SEQ ID NO.
  • SNP AD risk associated single nucleotide polymorphism
  • SNP1 is a portion of genomic nucleic acid sequence of plexin A4 (PLXNA4); (ii) SNP2 genotype T/T or T/C (or A/A or A/C in the complement) of SEQ ID NO: 1, wherein SNP2 is position 132,006,366 of SEQ ID NO: 1 identified by rsl0236235, wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; (iii) SNP3 genotype C/C or C/A (or G/G or G/T in the complement) of SEQ ID NO: 1 , wherein SNP3 identified by rsl 1761937 on SEQ ID NO: 1, wherein the SEQ ID NO.
  • PLXNA4 plexin A4
  • TSl PLXNA4 inhibitory agent 1 is a portionof genomic nucleic acid sequence of PLXNA4; and (iv) any combinations thereof, a therapeutically effective amount of a TSl PLXNA4 inhibitory agent.
  • a method for inhibiting or reducing tau phosphorylation in the brain comprising administering to a subject in need thereof a therapeutically effective amount of a TSl PLXNA4 inhibitory agent.
  • a method for inhibiting or reducing tau phosphorylation in the brain of a subject in need thereof comprising administering to a subject determined to have one or more of AD risk associated single nucleotide polymorphism (SNP) selected from: (i) SNP1 genotype A/A or A/C (or T/T or T/G in the complement) of SEQ ID NO: 1, wherein SNP1 is identified by rs277472 on SEQ ID NO: 1, wherein SEQ ID NO.
  • SNP AD risk associated single nucleotide polymorphism
  • SNP2 genotype T/T or T/C (or A/A or A/C in the complement) of SEQ ID NO: 1, wherein SNP2 is position 132,006,366 of SEQ ID NO: 1 identified by rsl0236235, wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4;
  • SNP3 genotype C/C or C/A or G/G or G/T in the complement
  • SNP3 identified by rsl 1761937 on SEQ ID NO: 1 wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; and (iv) any
  • a therapeutically effective amount of a TSl PLXNA4 inhibitory agent e.g., a TSl PLXNA4 inhibitory agent.
  • a method for treating a subject having or at risk for Alzheimer's disease comprising administering a therapeutically effective amount of a TSl PLXNA4 inhibitory agent to a subject in need thereof.
  • a method for treating a subject having or at risk for Alzheimer's disease comprising administering to a subject determined to have one or more of AD risk associated single nucleotide polymorphism (SNP) selected from: (i) SNP1 genotype A/A or A/C (or T/T or T/G in the complement) of SEQ ID NO: 1, wherein SNP1 is identified by rs277472 on SEQ ID NO: 1, wherein SEQ ID NO.
  • SNP AD risk associated single nucleotide polymorphism
  • SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of plexin A4 (PLXNA4); (ii) SNP2 genotype T/T or T/C (or A/A or A/C in the complement) of SEQ ID NO: 1, wherein SNP2 is position 132,006,366 of SEQ ID NO: 1 identified by rsl0236235, wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; (iii) SNP3 genotype C/C or C/A (or G/G or G/T in the complement) of SEQ ID NO: 1, wherein SNP3 identified by rsl 1761937 on SEQ ID NO: 1, wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; and (iv) any combinations thereof, a
  • a TS1 PLXNA4 inhibitory agent a TS1 PLXNA4 inhibitory agent
  • AD risk associated single nucleotide polymorphism selected from: (i) SNPl genotype A/A or A/C (or T/T or T/G in the complement) of SEQ ID NO: 1, wherein SNPl is identified by rs277472 on SEQ ID NO: 1, wherein SEQ ID NO.
  • SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of plexin A4 (PLXNA4); (ii) SNP2 genotype T/T or T/C (or A/A or A/C in the complement) of SEQ ID NO: 1, wherein SNP2 is position 132,006,366 of SEQ ID NO: 1 identified by rsl0236235, wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; (iii) SNP3 genotype C/C or C/A (or G/G or G/T in the complement) of SEQ ID NO: 1, wherein SNP3 identified by rsl 1761937 on SEQ ID NO: 1, wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; and (iv) any combinations thereof.
  • SNP single nucleotide polymorphism
  • SNP2 genotype T/T or T/C or A/A or A/C in the complement
  • SNP2 is position 132,006,366 of SEQ ID NO: 1 identified by rsl0236235, wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4
  • SNP3 genotype C/C or C/A or G/G or G/T in the complement
  • SNP3 identified by rsl 1761937 on SEQ ID NO: 1 wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4.
  • TS 1 PLXNA4 inhibitory agent is selected from the group consisting of small molecules, nucleic acids, nucleic acid analogues, peptides, proteins, antibodies, antigen binding fragments of antibodies, and any combinations thereof.
  • TS1 PLXNA4 inhibitory agent is an anti-miPv, antagomir, antisense oligonucleotide, ribozyme, aptamer, siRNA, shRNA, or RNAi agent.
  • TS1 PLXNA4 inhibitory agent does not bind or inhibit TS2 PLXNA4 or TS3 PLXNA4.
  • any of paragraphsl-15 further comprising assaying a biological sample from the subject before onset of said administering, wherein said assaying comprising measuring the absence of presence of a SNP selected from the group consisting of: (i) SNP1 genotype A/A or A/C (or T/T or T/G in the complement) of SEQ ID NO: 1, wherein SNP1 is identified by rs277472 on SEQ ID NO: 1, wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of plexin A4
  • PLXNA4 SNP2 genotype T/T or T/C (or A/A or A/C in the complement) of SEQ ID NO: 1, wherein SNP2 is position 132,006,366 of SEQ ID NO: 1 identified by rs 10236235, wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; (iii) SNP3 genotype C/C or C/A (or G/G or G/T in the complement) of SEQ ID NO: 1, wherein SNP3 identified by rsl 1761937 on SEQ ID NO: 1, wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; and (iv) any combinations thereof, wherein presence of one or more of SNP1-SNP3 is indicative of proceeding with said administering regimen.
  • said assaying comprises: subjecting the biological sample from a subject to at least one genotyping assay that determines the genotypes of at least one (e.g., one, two, or three) loci, wherein said loci are selected from: (i) SNP1, wherein SNP1 is identified by rs277472 on SEQ ID NO: 1, wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of plexin A4
  • PLXNA4 (ii) SNP2, wherein SNP2 is position 132,006,366 of SEQ ID NO: 1 identified by rsl 0236235, wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; (iii) SNP3, wherein SNP3 identified by rsl 1761937 on SEQ ID NO: 1, wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; and (iv) any combinations thereof.
  • SNP3 is a portion of genomic nucleic acid sequence of PLXNA4;
  • SNP3 identified by rsl 1761937 on SEQ ID NO: 1, wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; and (iv) any combinations thereof; and b.
  • SNP1 genotype A/A or A/C (or T/T or T/G in the complement) of SEQ ID NO: 1
  • SNP2 genotype T/T or T/C or A/A or A/C in the complement
  • SNP3 genotype C/C or C/A or G/G or G/T in the complement
  • SEQ ID NO: 1 any combinations thereof.
  • step (a) are further selected from: (i) SNP4, wherein SNP4 is identified by rsl593222 of SEQ ID NO: 1, wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; (ii) SNP5, wherein SNP5 is identified by rs6959579 of SEQ ID NO: 1, wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; (iii) SNP6, wherein SNP6 is identified by rsl7166339 of SEQ ID NO: 1, wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4.
  • An assay comprising:
  • SNP3 is a portion of genomic nucleic acid sequence of PLXNA4;
  • SNP3 identified by rsl 1761937 on SEQ ID NO: 1, wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; and (iv) any combinations thereof;
  • the at least one AD risk associated SNP is selected from: (i) SNP1 genotype A/A or A/C (or T/T or T/G in the complement) of SEQ ID NO: 1, (ii) SNP2 genotype T/T or T/C (or A/A or A/C in the complement) of SEQ ID NO: 1, (iii) SNP3 genotype C/C or C/A (or G/G or G/T in the complement) of SEQ ID NO: 1, and (iv) any
  • An assay comprising: a. contacting a biological sample obtained from a subject with an allele specific detectable oligonucleotide specific for at least one of the following SNPs: (i) SNP1 genotype A/A or A/C (or T/T or T/G in the complement) of SEQ ID NO: 1, (ii) SNP2 genotype T/T or T/C (or A/A or A/C in the complement) of SEQ ID NO: 1, (iii) SNP3 genotype C/C or C/A (or G/G or G/T in the complement) of SEQ ID NO: 1, and (iv) any combinations thereof;
  • SNP3 is a portion of genomic nucleic acid sequence of PLXNA4;
  • SNP3 identified by rsl 1761937 on SEQ ID NO: 1, wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; and (iv) any combinations thereof;
  • SNP3 is a portion of genomic nucleic acid sequence of PLXNA4;
  • SNP3 identified by rsl 1761937 on SEQ ID NO: 1, wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; and (iv) any combinations thereof;
  • At least one AD risk associated SNP is selected from: (i) SNP1 genotype A/A or A/C (or T/T or T/G in the complement) of SEQ ID NO: 1, (ii) SNP2 genotype T/T or T/C (or A/A or A/C in the complement) of SEQ ID NO: 1, (iii) SNP3 genotype C/C or C/A (or G/G or G/T in the complement) of SEQ ID NO: 1, and (iv) any combinations thereof; and
  • step (b) identifying the subject as having an increased probability of having AD when at least one AD risk associated SNP is detected in step (b).
  • An assay for identifying a subject having or at risk for Alzheimer's disease comprising:
  • An assay for determining if a subject is in need of treatment or prevention for Alzheimer's disease comprising: a. subjecting a test sample from a subject to at least one genotyping assay that determines the genotypes of at least one (e.g., one, two, or three) loci, wherein said loci are selected from: (i) SNP1, wherein SNP1 is identified by rs277472 on SEQ ID NO: 1, wherein SEQ ID NO.
  • SNP1 is a portion of genomic nucleic acid sequence of plexin A4 (PLXNA4);
  • SNP2 is position 132,006,366 of SEQ ID NO: 1 identified by rsl0236235, wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4;
  • SNP3 is a portion of genomic nucleic acid sequence of PLXNA4; and
  • Alzheimer's disease comprising:
  • the at least one AD risk associated SNP is selected from: (i) SNP1 genotype A/A or A/C (or T/T or T/G in the complement) of SEQ ID NO: 1, (ii) SNP2 genotype T/T or T/C (or A/A or A/C in the complement) of SEQ ID NO: 1, (iii) SNP3 genotype C/C or C/A (or G/G or G/T in the complement) of SEQ ID NO: 1, and (iv) any
  • step (b) identifying or selecting the subject for treatment or prevention for AD when at least one AD risk associated SNP is detected in step (b).
  • Alzheimer's disease comprising:
  • step (a) comparing the measured intensity of the signal with a reference value and if the measured intensity is increased relative to the reference value; and e. identifying or selecting the subject for treatment or prevention for AD when at least one SNP of step (a) is detected.
  • An assay for selecting a subject having or at risk for Alzheimer's disease, wherein subject is susceptible to treatment with a TS1 PLXNA4 inhibitory agent comprising:
  • SNP3 is a portion of genomic nucleic acid sequence of PLXNA4;
  • SNP3 identified by rsl 1761937 on SEQ ID NO: 1, wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; and (iv) any combinations thereof;
  • An assay for selecting a subject having or at risk for Alzheimer's disease, wherein subject is susceptible to treatment with a TS1 PLXNA4 inhibitory agent comprising:
  • SNP3 is a portion of genomic nucleic acid sequence of PLXNA4;
  • SNP3 identified by rsl 1761937 on SEQ ID NO: 1, wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; and (iv) any combinations thereof;
  • the at least one AD risk associated SNP is selected from: (i) SNP1 genotype A/A or A/C (or T/T or T/G in the complement) of SEQ ID NO: 1, (ii) SNP2 genotype T/T or T/C (or A/A or A/C in the complement) of SEQ ID NO: 1, (iii) SNP3 genotype C/C or C/A (or G/G or G/T in the complement) of SEQ ID NO: 1, and (iv) any
  • step (b) identifying Alzheimer's disease in the subject as susceptible for treatment with a TS 1 PLXNA4 inhibitory agent when at least one AD risk associated SNP is detected in step (b).
  • An assay for selecting a subject having or at risk for Alzheimer's disease, wherein subject is susceptible to treatment with a TS1 PLXNA4 inhibitory agent comprising:
  • step (a) comparing the measured intensity of the signal with a reference value and if the measured intensity is increased relative to the reference value; and e. identifying Alzheimer's disease in the subject as susceptible for treatment with a TS 1 PLXNA4 inhibitory agent when at least one SNP of step (a) is detected. assay comprising:
  • identifying the subject as having an increased probability of having AD assay comprising:
  • An assay for identifying a subject having or at risk for Alzheimer's disease comprising:
  • TS1 and/or TS3 PLXNA4 transcript is increased relative to a reference value.
  • An assay for determining an increased risk of a subject for developing Alzheimer's disease comprising
  • An assay for determining an increased risk of a subject for developing Alzheimer's disease comprising:
  • PLXNA4 transcript is increased relative to a reference value.
  • An assay for selecting a subject having or at risk for Alzheimer's disease, wherein subject is susceptible to treatment with a TSl PLXNA4 inhibitory agent comprising:
  • An assay for identifying a subject having or at risk for Alzheimer's disease comprising:
  • a measuring or quantifying the level of expression or amount of TSl or TS3 PLXNA4 transcript in a first biological sample obtained from a subject; and b. measuring or quantifying the level of expression level or amount of TSl or TS3 PLXNA4 transcript in a second biological sample obtained from a subject;
  • Tsl or TS3 PLXNA4 transcript is increased in the second biological sample relative to the first biological sample by at least 20%.
  • PLXNA4 transcript in a biological sample obtained from a subject a biological sample obtained from a subject; and b. identifying the subject as having or at risk for Alzheimer's disease if the
  • TS3 PLXNA4 transcript amount of TS3 PLXNA4 transcript, TS1 PLXNA4 transcript, or both are increased relative to a reference TS3 PLXNA4 transcript value.
  • a determination module configured to identify and detect at least one single nucleotide polymorphism (SNP) in a biological sample of a subject, wherein the SNP is selected from: (i) SNP1, wherein SNP1 is identified by rs277472 on SEQ ID NO: 1, wherein SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of plexin A4 (PLXNA4); (ii) SNP2, wherein SNP2 is position 132,006,366 of SEQ ID NO: 1 identified by rsl0236235, wherein SEQ ID NO.
  • SNP single nucleotide polymorphism
  • SNP3 is a portion of genomic nucleic acid sequence of PLXNA4;
  • SNP3 identified by rsl 1761937 on SEQ ID NO: 1, wherein the SEQ ID NO. 1 is a portion of genomic nucleic acid sequence of PLXNA4; and (iv) any combinations thereof;
  • a storage module configured to store output data from the determination
  • a computing module adapted to identify from the output data at least one of AD risk associated SNP is present in the output data stored on the storage module, wherein the AD risk associated SNP is selected from: (i) SNP1 genotype A/A or A/C (or T/T or T/G in the complement) of SEQ ID NO: 1 , (ii) SNP2 genotype T/T or T/C (or A/A or A/C in the complement) of SEQ ID NO: 1, (iii) SNP3 genotype C/C or C/A (or G/G or G/T in the complement) of SEQ ID NO: 1, and (iv) any combinations thereof; and
  • a display module for displaying if any of the AD risk associated SNP was identified or not, and/or displaying the detected alleles.
  • SEQ ID NO: 1 refers to the genomic sequence of human PLXNA4 of Gene ID 91584, which can be found at bases 131808091 to 132333447 of the human chromosome 7 NCBI Reference Sequence: NC 000007.13.
  • TS 1 PLXNA4 transcript or “TS 1 " refers to the full-length Plexin A4 (PLXNA4) transcript that contains 31 exons and encodes an isoform with 1,894 residues.
  • TS2 PLXNA4 transcript or “TS2” and “TS3 PLXNA4 transcript” or “TS3” refer to two alternatively spliced transcripts each of which contains three exons, thereby yielding shorter isoforms of 522 residues (TS2) and 492 residues (TS3), respectively.
  • treatment means preventing the progression of the disease, or altering the course of the disorder (for example, but are not limited to, slowing the progression of the disorder), or reversing a symptom of the disorder or reducing one or more symptoms and/or one or more biochemical markers in a subject, preventing one or more symptoms from worsening or progressing, promoting recovery or improving prognosis.
  • nucleic acid or “oligonucleotide” or “polynucleotide” refers to a polymer or an oligomer of nucleotide or nucleoside monomers consisting of nucleobases, sugars and intersugar linkages.
  • oligonucleotide also includes polymers or oligomers comprising non-naturally occurring monomers, or portions thereof, which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases.
  • administering includes any statistically significant improvement in one or more indicia of the disease or disorder.
  • the terms “prevent,” “preventing” and “prevention” refer to the avoidance or delay in manifestation of one or more symptoms or measurable markers of a disease or disorder, e.g., Alzheimer's disease.
  • a delay in the manifestation of a symptom or marker is a delay relative to the time at which such symptom or marker manifests in a control or untreated subject with a similar likelihood or susceptibility of developing the disease or disorder.
  • prevent include not only the avoidance or prevention of a symptom or marker of the disease, but also a reduced severity or degree of any one of the symptoms or markers of the disease, relative to those symptoms or markers in a control or non-treated individual with a similar likelihood or susceptibility of developing the disease or disorder, or relative to symptoms or markers likely to arise based on historical or statistical measures of populations affected by the disease or disorder.
  • reduced severity is meant at least a 10% reduction in the severity or degree of a symptom or measurable disease marker, relative to a control or reference, e.g., at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or even 100% (i.e., no symptoms or measurable markers).
  • the oligonucleotide can be single-stranded or double-stranded.
  • a single-stranded oligonucleotide can have double-stranded regions and a double-stranded oligonucleotide can have single-stranded regions.
  • the oligonucleotide can have a hairpin structure or have a dumbbell structure.
  • the oligonucleotide can be circular, e.g., wherein the 5 'end of the oligonucleotide is linked to the 3' end of the oligonucleotide.
  • the depiction of a single strand also defines the sequence of the complementary strand.
  • a nucleic acid also encompasses the complementary strand of a depicted single strand.
  • many variants of a nucleic acid can be used for the same purpose as a given nucleic acid.
  • a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.
  • a single strand provides a probe that can hybridize to the target sequence under stringent hybridization conditions.
  • a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.
  • the oligonucleotides described herein can comprise any oligonucleotide modification described herein and below.
  • the oligonucleotide comprises at least one modification.
  • the modification is selected from the group consisting of a sugar modification, a non-phosphodiester inter-sugar (or inter- nucleoside) linkage, nucleobase modification, and ligand conjugation.
  • an oligonucleotide can be of any length. In some embodiments, oligonucleotides can range from about 6 to 100 nucleotides in length. In various related embodiments, the oligonucleotide can range in length from about 10 to about 50 nucleotides, from about 10 to about 35 nucleotides, from about 15 to about 30 nucleotides, from about 20 to about 30 nucleotides in length. In some embodiments, oligonucleotide is from about 8 to about 39 nucleotides in length.
  • the oligonucleotide is 6 to 25 nucleotides in length (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, or 24 nucleotides in length). In some embodiments the oligonucleotide is 25-30 nucleotides. In some embodiments, the single-stranded oligonucleotide is 15 to 29 nucleotides in length. In some other embodiments, the oligonucleotide is from about 18 to about 25 nucleotides in length. In some embodiments, the oligonucleotide is about 23 nucleotides in length. In some embodiments, a single-stranded oligonucleotide is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 17, 18, or 19 nucleotides in length.
  • the oligonucleotide can be completely DNA, completely R A, or comprise both R A and DNA nucleotides. It is to be understood that when the oligonucleotide is completely DNA, RNA, or a mix of both, the oligonucleotide can comprise one or more oligonucleotide modifications described herein.
  • the oligonucleotides can include one or more oligonucleotide or nucleic acid modifications. Unmodified oligonucleotides can be less than optimal in some applications, e.g., unmodified
  • oligonucleotides can be prone to degradation by e.g., cellular nucleases.
  • chemical modifications to one or more of the subunits of oligonucleotide can confer improved properties, e.g., can render oligonucleotides more stable to nucleases.
  • oligonucleotide modifications can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester intersugar linkage; (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar; (iii) wholesale replacement of the phosphate moiety with "dephospho" linkers; (iv) modification or replacement of a naturally occurring base with a non-natural base; (v) replacement or modification of the ribose-phosphate backbone, e.g.
  • PNA peptide nucleic acid
  • modification of the 3' end or 5' end of the oligonucleotide e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, e.g., conjugation of a ligand, to either the 3' or 5' end of oligonucleotide
  • modification of the sugar e.g., six membered rings.
  • a modification described herein can be the sole modification, or the sole type of modification included on multiple nucleotides, or a modification can be combined with one or more other modifications described herein.
  • the modifications described herein can also be combined onto an oligonucleotide, e.g. different nucleotides of an oligonucleotide have different modifications described herein.
  • the phosphate group in the intersugar linkage can be modified by replacing one of the oxygens with a different substituent.
  • One result of this modification to RNA phosphate intersugar linkages can be increased resistance of the oligonucleotide to nucleolytic breakdown.
  • modified phosphate groups include phosphorothioate,
  • Some exemplary intersugar linkage modifications include phosphonate phosphorothioate, phosphorodithioate, phosphoramidate methoxyethyl phosphoramidate, formacetal, thioformacetal,
  • An oligonucleotide can include modification of all or some of the sugar groups of the nucleic acid.
  • the 2' position H, DNA;or OH, RNA
  • enhanced stability is expected since the 2'-hydroxyl can no longer be deprotonated to form a 2'-alkoxide ion.
  • the 2'-alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom.
  • Preferred sugar modifications are 2'-O-Me (2'-O-methyl), -O- MOE (2'-O-methoxyethyl), 2'-F, 2'-O-[2-(methylamino)-2-oxoethyl] (2'-0-NMA), T-S- methyl, 2'-0-CH 2 -(4'-C) (LNA), 2'-0-CH 2 CH 2 -(4'-C) (ENA), 2'-0-aminopropyl (2'-0-AP), 2 * -0-dimethylaminoethyl (2 * -0-DMAOE), 2 * -0-dimethylaminopropyl (2 * -0-DMAP), and 2 * - O-dimethylaminoethyloxy ethyl (2'-0-DMAEOE) .
  • nucleotide when a particular nucleotide is linked through its 2'- position to the next nucleotide, the sugar modifications described herein can be placed at the 3 '-position of the sugar for that particular nucleotide, e.g., the nucleotide that is linked through its 2' -position.
  • a modification at the 3 ' position can be present in the xylose configuration.
  • xylose configuration refers to the placement of a substituent on the C3 ' of ribose in the same configuration as the 3 ' -OH is in the xylose sugar.
  • Adenine, cytosine, guanine, thymine and uracil are the most common bases (or nucleobases) found in nucleic acids. These bases can be modified or replaced to provide oligonucleotides having improved properties. For example, nuclease resistant bases (or nucleobases) found in nucleic acids. These bases can be modified or replaced to provide oligonucleotides having improved properties. For example, nuclease resistant
  • oligonucleotides can be prepared with these bases or with synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and any one of the above modifications.
  • nucleobases e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine
  • substituted or modified analogs of any of the above bases and "universal bases” can be employed.
  • the nucleotide is said to comprise a modified nucleobase and/or a nucleobase modification herein. Modified nucleobase and/or nucleobase
  • conjugated moieties e.g. a ligand described herein.
  • Preferred conjugate moieties for conjugation with nucleobases include cationic amino groups which can be conjugated to the nucleobase via an appropriate alkyl, alkenyl or a linker with an amide linkage.
  • Modified nucleobases include other synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2-(halo)adenine, 2-(alkyl)adenine, 2- (propyl)adenine, 2-(amino)adenine, 2-(aminoalkyll)adenine, 2-(aminopropyl)adenine,
  • 3- (deaza)-5-(aza)cytosine 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5- (alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine,
  • 2,4-(dithio)uracil 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5- (allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(l ,3-diazole- 1 -alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil,
  • a universal nucleobase is any modified or nucleobase that can base pair with all of the four naturally occurring nucleobases without substantially affecting the melting behavior, recognition by intracellular enzymes or activity of the oligonucleotide duplex.
  • Some exemplary universal nucleobases include, but are not limited to, 2,4- difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6- methylbenzimidazle, 4-methylbenzimidazle, 3-methyl isocarbostyrilyl, 5- methyl
  • subject and “individual” are used interchangeably herein, and refer to an animal, for example a human, to whom treatment, including prophylactic treatment, with a pharmaceutical composition according to the present invention, is provided.
  • treatment including prophylactic treatment
  • pharmaceutical composition according to the present invention
  • subject refers to human and non-human animals.
  • non-human animals and “non-human mammals” are used interchangeably herein, and includes all vertebrates, e.g., mammals, such as non-human primates, (particularly higher primates), sheep, dog, rodent (e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, and non- mammals such as chickens, amphibians, reptiles etc.
  • the subject is human.
  • the subject is an experimental animal or animal substitute as a disease model. The term does not denote a particular age or sex.
  • adult and newborn subjects are intended to be covered.
  • subjects include humans, dogs, cats, cows, goats, and mice.
  • the term subject is further intended to include transgenic species.
  • the subject can be of European ancestry.
  • the subject can be of African American ancestry.
  • the subject can be of Asian ancestary.
  • compositions refers to a composition that usually contains an excipient, such as a pharmaceutically acceptable carrier that is conventional in the art and that is suitable for administration to cells or to a subject.
  • compositions for topical (e.g., oral mucosa, respiratory mucosa) and/or oral administration can be in the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, oral rinses, or powders, as known in the art and described herein.
  • the compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, University of the Sciences in Philadelphia (2005) Remington: The Science and Practice of Pharmacy with Facts and Comparisons, 21st Ed.
  • the terms “increased”, “increase” or “enhance” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%>, or at least about 80%>, or at least about 90%> or up to and including a 100% increase or any increase between 10-100% as compared to a reference value or level, or at least about a 1.5-fold, at least about a 1.6-fold, at least about a 1.7-fold, at least about a 1.8- fold, at least about a 1.9-fold, at least about a 2-fold, at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold, at least about a 10-fold increase, any increase between 2-fold and 10-fold, at least about a 25-fold increase, or greater as compared to a reference level.
  • an increase is at least one standard deviation greater than, or at least two standard deviations, or more, greater than a median or mean reference level.
  • median or mean reference levels can be obtained, for example, from five or more samples obtained from subjects not having Alzheimer's disease, or from five or more samples obtained from the same subject at different timepoints.
  • the reference level can be obtained or measured in a reference biological sample, such as a reference sample obtained from an age-matched normal control (e.g., an age-matched subject not having Alzheimer's disease), or a reference sample from the same subject at an earlier timpoint, for example, a "first biological sample.”
  • a "reference value” is thus, in some embodiments, a predetermined reference level, such as an average or median amount or level of TS3 or TS1 PLXNA4 transcript obtained from, for example, biological samples from a population of healthy subjects that are in the chronological age group matched with the chronological age of the tested subject.
  • the reference can be a normal healthy subject with no genetic susceptibility for AD.
  • a normal healthy subject is not a carrier of any of the late onset AD risk associated alleles described herein or is not diagnosed with any forms of AD such as early-onset autosomal-dominant AD, or any neurodegenerative disorders.
  • the reference can be also a control sample, a pooled sample of control individuals or a numeric value or range of values based on the same.
  • biological sample or “subject sample” or “sample” refer to a quantity of tissue or fluid, or a cell or population of cells obtained from a subject.
  • the biological sample is a blood sample, including, for example, a serum sample, or a plasma sample. Most often, the sample has been removed from a subject, but the term “biological sample” can also, in some embodiments, refer to cells or tissue or a quantity of tissue or fluid analyzed in vivo, i.e. without removal from the subject.
  • a biological sample or tissue sample includes, but is not limited to, blood, plasma, serum, cerebrospinal fluid, lymph fluid, bone marrow, tumor biopsy, urine, stool, sputum, pleural fluid, nipple aspirates, lymph fluid, the external sections of the skin, lung tissue, adipose tissue, connective tissue, sub-epithelial tissue, epithelial tissue, liver tissue, kidney tissue, uterine tissue, respiratory tissues, breast tissue, gastrointestinal tissue, and genitourinary tract tissue, tears, saliva, milk, cells (including, but not limited to, blood cells), biopsies, scrapes (e.g., buccal scrapes), tumors, organs, and also samples of an in vitro cell culture constituent.
  • a "biological sample” can comprise cells from the subject, but the term can also refer to non-cellular biological material, such as non-cellular fractions of blood, saliva, or urine.
  • the term “reduced” or “reduce” or “decrease” as used herein generally means a decrease by a statistically significant amount relative to a reference. However, for avoidance of doubt, “reduced” means statistically significant decrease of at least 10% as compared to a reference level, for example a decrease by at least 20%, at least 30%, at least 40%, at least t 50%), or least 60%>, or least 70%>, or least 80%>, at least 90%> or more, up to and including a 100%) decrease (i.e. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level, as that term is defined herein.
  • the term "consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • Alzheimer's disease is the most frequent age-related dementia affecting 5.4 million Americans including 13% of people ages 65 and older and over 40%> of people ages 85 and older. 1 Genetic factors account for much of the risk for developing AD with heritability estimates between 60% and 80%. 2
  • the apolipoprotein E (APOE) ⁇ 4 allele is a well-recognized major risk factor for late onset AD, increasing the odds of disease in a dose-dependent fashion.
  • APOE apolipoprotein E
  • 3 Common polymorphisms in ten additional genes have been robustly established as risk factors for AD using large-scale genome-wide association studies (GWAS) and meta-analyses. 4 ' 5 These polymorphisms link to mechanisms of AO metabolism, lipid metabolism, inflammation, and axon guidance.
  • the heritability of AD explained by APOE is about 17.5% and by each of the novel GWAS loci is less than 1%, suggesting that less than 30% of the genetic contribution to AD is explained by known common polymorphisms. 4 ' 8 The remaining heritability may be due to additional common variants of weaker effect, rare variants, copy- number variants, insertion-deletion polymorphisms, and gene-gene and gene-environment
  • hyperphosphorylation of tau protein a terminal step leading to breakdown of neuronal signaling and microtubule formation.
  • Subjects The GWAS was performed using a community-based sample consisting of 61 incident AD cases and 2,530 cognitively normal controls from 1,232 families in the Framingham Heart Study (FHS). The top findings were evaluated in a second cohort including 1,819 AD cases and 1,969 controls from 2,265 families containing multiple members affected by AD in the National Institute on Aging - Late Onset Alzheimer Disease (NIA-LOAD) Study. Clinical, demographic, pedigree and genetic data for both cohorts were obtained from dbGaP on the worldwide web at www.ncbi.nlm.nih.gov/gap. Additional details about the ascertainment, evaluation and characteristics of these subjects are provided.
  • FHS Framingham Heart Study
  • Amyloid Precursor Protein APP
  • Amyloid ⁇ ⁇
  • tau Analyses Methods for investigating the effect of PLXNA4 on the processing of APP, ⁇ and tau protein are presented.
  • the mean onset age of AD among the 61 incident cases in the FHS dataset was about 10 years older than that for the 1,819 AD cases in the NIA-LOAD datbase.
  • the frequency of the APOE ⁇ 4 allele in unaffected subjects was comparable between the two family datasets, but the ⁇ 4/ ⁇ 4 genotype frequency was approximately nine times greater in AD subjects in the NIA-LOAD cohort compared to AD subjects in the FHS cohort.
  • the proportion of AD subjects lacking ⁇ 4 was about three times larger in the FHS cohort.
  • PLXNA4 is a member of a family of receptors for transmembrane, secreted and GPI-anchored semaphorins in vertebrates 14 and is a receptor for secreted semaphorin class 3 (SEMA3A) and class 6 (SEMA6) proteins which play an important role in semaphorin signaling and axon guidance.
  • SEMA3A secreted semaphorin class 3
  • SEMA6 class 6
  • TS1 PLXNA4 transcript is the full-length PLXNA4 transcript that contains 31 exons and encodes an isoform with 1,894 residues.
  • TS2 PLXNA4 transcript or “TS2” and “TS3 PLXNA4 transcript” or “TS3” refere to two alternatively spliced transcripts each of which contains three exons yielding shorter isoforms of 522 residues (TS2) and 492 residues (TS3), respectively.
  • TS2 PLXNA4 transcript or “TS2” and “TS3 PLXNA4 transcript” or “TS3” refere to two alternatively spliced transcripts each of which contains three exons yielding shorter isoforms of 522 residues (TS2) and 492 residues (TS3), respectively.
  • TS2 PLXNA4 transcript or “TS2”
  • IRE motif was identified in the sequence surrounding the most significantly associated SNP in the combined dataset (rs 10236235) but only in the presence of the protective allele T(FIG. IE).
  • Other AD-associated SNPs were also predicted to impact splicing. Bioinformatic evaluation confirmed that the longer isoform contains a transmembrane domain, while the shorter forms are predicted as secreted forms (FIG. IF), indicating that different isoforms can have distinct functional consequences related to AD.
  • Semaphorin-3A SEMA3A-FC. Immunoblotting with AT8 (anti-phospho-Tau at
  • PLXNA4 is a member of a family of receptors for transmembrane, secreted and GPI-anchored semaphorins in vertebrates 14 and is a receptor for secreted semaphorin class 3 (SEMA3A) and class 6 (SEMA6) proteins which play an important role in semaphorin signaling and axon guidance.
  • SEMA3A secreted semaphorin class 3
  • SEMA6 class 6
  • pathogenesis specifically through tau phosphorylation leading to tangle formation and neuronal death.
  • TS1 and TS3 There are three known alternatively spliced PLXNA4 transcripts.
  • the full-length transcript (TSl) contains 31 exons and encodes an isoform with 1,894 residues.
  • Two alternatively spliced transcripts each contain three exons yielding shorter isoforms of 522 residues (TS2) and 492 residues (TS3), respectively.
  • TS2 522 residues
  • TS3 492 residues
  • the distinct PLXNA 4 association peaks in the discovery and replication datasets flank an exon that is present only in TS3.
  • Our bioinformatic analysis identified predicted intronic splicing regulatory elements near the most strongly associated SNP in each dataset. However, our finding of increased expression of both TS1 and TS3 indicate that the genetic mechanism can also involve transcription regulatory elements.
  • a direct link of PLXNA4 expression to AD is supported by the evidence described herein of increased expression of TS1 and TS3 isoforms in post-mortem neuronal tissue from AD cases compared to controls.
  • the findings that the relative increase of TS1 is much greater than TS3 in AD cases, and TS1 expression is significantly correlated with clinical and neuropathological severity measures of AD are consistent with our observation of increased phosphorylation of tau by SEMA3 A bound specifically to TS 1.
  • our findings in sera from a group of young controls suggest that differential expression of these isoforms may be genetically regulated beginning in early life.
  • U we applied a novel approach to address the differences between the FHS and NIA-LOAD datasets.
  • the minor allele C for rsl2539196 decreased the liability rank by 0.09 in FHS and accounts for a 15% reduction in the propensity rank in NIA-LOAD (Table 2).
  • Further scrutiny of the PLXNA4 findings revealed that the most significant SNPs in each dataset are clustered in two distinct regions approximately 78,240 base pairs apart in intron 2 of the largest transcript (TS1) and flank an alternatively spliced exon present only in a much shorter transcript (TS3) (Fig 4D).
  • MS4A4/MS4A6E, CD2AP, CD33 and EPHA1 are associated with late-onset
  • MS4A6A/MS4A4E, EPHA1 , CD33 and CD2AP are associated with Alzheimer's disease. Nat Genet 201 1 ; 43 :429435
  • Plexins are a large family of receptors for transmembrane, secreted, and GPI-anchored semaphorins in vertebrates. Cell 1999; 99:71-80 Suto F, Ito K, Uemura M, et al. Plexin-a4 mediates axon-repulsive activities of both secreted and transmembrane semaphorins and plays roles in nerve fiber guidance. J Neurosci 2005; 25:3628-3637
  • CH chromosome
  • POS base pair position from build 37
  • MA minor allele
  • MAF minor allele frequency
  • ⁇ estimate from the regression model
  • P P value in each study or in metaanalysis
  • Table 2 SNP -based and gene-based association results with genotyped and imputed SNPs for AD risk in the FHS dataset and for AD propensity score in the NIA-LOAD dataset.
  • CH chromosome
  • POS base pair position from build 37
  • RA risk allele
  • nSNPs number of SNPs tested in gene -based analysis
  • RAF risk allele frequency
  • ⁇ estimate from the regression model
  • P P value
  • Z Zscore in meta-analysis weighted by the number of SNPs in a gene
  • NA not applicable.
  • Table 3 PLXNA4 isoform levels in brain from neuropatho logically examined AD cases and controls
  • TS1 isoform 1
  • TS3 isoform 3
  • SE standard error
  • * T-tests account for unequal variances

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Abstract

La présente invention se rapporte à des procédés, à des dosages et à des systèmes permettant de détecter un risque accru de la maladie d'Alzheimer chez un sujet par identification d'au moins un polymorphisme d'acide nucléique décrit ici dans un échantillon biologique prélevé sur le sujet. Les niveaux des gènes associés au polymorphisme d'acide nucléique décrit ici sont également déterminés pour permettre la détection d'un risque plus élevé de la maladie d'Alzheimer. La présente invention porte en outre sur des procédés permettant de traiter la maladie d'Alzheimer par administration à un sujet qui en a besoin d'un agent inhibiteur TS1 PLXNA4.
PCT/US2014/037479 2013-05-09 2014-05-09 Utilisation de la plexine-a4 comme biomarqueur et cible thérapeutique pour la maladie d'alzheimer WO2014183023A1 (fr)

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